Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system

ABSTRACT

This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. The invention provides a microsystem platform and a micromanipulation device for manipulating the platform that utilizes the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels. The microsystem, platforms of the invention are also optionally provided having system informatics and data acquisition, analysis and storage and retrieval informatics encoded on the surface of the disk opposite to the surface containing the fluidic components. Methods specific for the apparatus of the invention for performing any of a wide variety of microanalytical or microsynthetic processes are provided.

This application is a divisional of U.S. Ser. No. 08/768,990, filed Dec.18, 1996, now U.S. Pat. No. 6,319,469, issued Nov. 20, 2001, whichapplication claims priority to U.S. patent application, Ser. No.08/761,063, filed Dec. 5, 1996 (Attorney Docket No. 95,1408-D) and toU.S. Provisional Applications, Ser. No. 60/008,819, filed Dec. 18, 1995,and 60/023,756, filed Aug. 12, 1996, the disclosures of each of whichare explicitly incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus for performingmicroanalytic and microsynthetic analyses and procedures. In particular,the invention relates to microminiaturization of genetic, biochemicaland chemical processes related to analysis, synthesis and purification.Specifically, the invention provides a microsystem platform and amicromanipulation device to manipulate the platform by rotation, therebyutilizing the centripetal forces resulting from rotation of the platformto motivate fluid movement through microchannels embedded in themicroplatform. The microsystem platforms of the invention are alsoprovided optionally having system informatics and data acquisition,analysis and storage and retrieval informatics encoded on the surface ofthe disk opposite to the surface containing the fluidic components.Methods for performing any of a wide variety of microanalytical ormicrosynthetic processes using the microsystems apparatus of theinvention are also provided.

2. Background of the Related Art

In the field of medical, biological and chemical assays, a mechanicaland automated fluid handling systems and instruments produced to operateon a macroscopic (i.e., milliliters and milligrams) scale are known inthe prior art.

U.S. Pat. No. 4,279,862, issued Jul. 21, 1981 to Bertaudiere et al.disclose a centrifugal photometric analyzer.

U.S. Pat. No. 4,381,291, issued Apr. 26, 1983 to Ekins teach analyticmeasurement of free ligands.

U.S. Pat. No. 4,515,889, issued May 7, 1985 to Klose et al. teachautomated mixing and incubating reagents to perform analyticaldeterminations.

U.S. Pat. No. 4,676,952, issued June 30, 1987 to Edelnann et al teach aphotometric analysis apparatus.

U.S. Pat. No. 4,745,072, issued May 17, 1988 to Ekins disclosesimmunoassay in biological fluids.

U.S. Pat. No. 5,160,702 issued Nov. 3, 1992 to Kopf-Sill et al.discloses a centrifuge rotor for analyzing solids in a liquid.

U.S. Pat. No. 5,171,695, issued Dec. 15, 1992 to Ekins disclosesdetermination of analyte concentration using two labeling markers.

U.S. Pat. No. 5,173,262 issued Dec. 22, 1996 to Burtis et al. disclosesa centrifuge rotor for processing liquids.

U.S. Pat. No. 5,242,803, issued Sep. 7, 1993 to Burtis et al. disclose arotor assembly for carrying out an assay.

U.S. Pat. No. 5,409,665, issued Apr. 25, 1995 to Burd disclose cuvettefilling in a centrifuge rotor.

U.S. Pat. No. 5,413,732, issued May 9, 1995 to Buhl et al. teachpreparation of lyophilized reagent spheres for use in automatedcentrifugal blood analyzers.

U.S. Pat. No. 5,432,009, issued Jul. 11, 1995 to Ekins discloses amethod for analyzing analytes in a liquid.

U.S. Pat. No. 5,472,603 issued Dec. 5, 1995 to Schembri discloses ananalytical rotor for performing fluid separations.

Anderson, 1968, Anal. Biochem. 28: 545-562 teach a multiple cuvetterotor for cell fractionation.

Renoe et al., Clin. Chem. 20: 955-960 teach a “minidisc” module for acentrifugal analyzer.

Burtis et al., Clin. Chem. 20: 932-941 teach a method for dynamicintroduction of liquids into a centrifugal analyzer.

Fritsche et al. 1975, Clin. Biochem. 8: 240-246 teach enzymatic analysisof blood sugar levels using a centrifugal analyzer.

Burtis et al., Clin. Chem. 21: 1225-1233 a multipurpose optical systemfor use with a centrifugal analyzer.

Hadjiioannou et al. 1976, Clin. Chem. 22: 802-805 teach automatedenzymatic ethanol determination in biological fluids using a miniaturecentrifugal analyzer.

Lee et al., 1978, Clin. Chem. 24: 1361-1365 teach an automated bloodfractionation system.

Cho et al., 1982, Clin. Chem. 28: 1961-1965 teach a multichannelelectrochemical centrifugal analyzer.

Bertrand et al., 1982, Clinica Chimica Acta 119: 275-284 teach automateddetermination of serum 5′-nucleotidase using a centrifugal analyzer.

Schembri et al., 1992, Clin. Chem. 38: 1665-1670 teach a portable wholeblood analyzer.

Walters et al., 1995, Basic Medical Laboratorv Technoloaies, 3^(rd) ed.,Delmar Publishers: Boston teach a variety of automated medicallaboratory analytic techniques.

Recently, microanalytical devices for performing select reactionpathways have been developed.

U.S. Pat. No. 5,006,749, issued Apr. 9, 1991 to White disclose methodsand apparatus for using ultrasonic energy to move microminiatureelements.

U.S. Pat. No. 5,252,294, issued Oct. 12, 1993 to Kroy et al. teach amicromechanical structure for performing certain chemical microanalyses.

U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teachfluid handling on microscale analytical devices.

U.S. Pat. No. 5,368,704 issued Nov. 29, 1994 to Madou et al. teachmicroelectrochemical valves.

International Application, Publication No. WO93/22053, published 11 Nov.1993 to University of Pennsylvania disclose microfabricated detectionstructures.

International Application, Publication No. WO93/22058, published 11 Nov.1993 to University of Pennsylvania disclose microfabricated structuresfor performing polynucleotide amplification.

Columbus et al., 1987, Clin. Chem. 33: 1531-1537 teach fluid managementof biological fluids.

Ekins et al., 1992, Ann. Biol. Clin. 50: 337-353 teach a multianalyticalmicrospot immunoassay.

Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose manipulation offluids on straight channels micromachined into silicon.

The prior art discloses synthetic microchips for performingmicroanalytic and microsynthetic methods. One drawback in the prior artmicroanalytical methods and apparati has been the difficulty indesigning systems for moving fluids on the microchips through channelsand reservoirs having diameters in the 10-100 μm range. Also, thedevices disclosed in the prior art have required separate data analysisand storage media to be integrated into an instrument for performing themicroanalysis, thereby unnecessarily increasing the complexity of theinstruments designed to use the microchips; without a concomitantincrease in the flexibility or usefulness of these machines.

There remains a need for a simple, flexible, reliable, rapid andeconomical microanalytic and microsynthetic reaction platform forperforming biological, biochemical and chemical analyses and synthesesthat can move fluids within the structural components of a microsystemsplatform. Such a platform should be able to move nanoliter to microliteramounts of fluid, including reagents and reactants, at rapid rates toeffect the proper mixing of reaction components, removal of reactionside products, and isolation of desired reaction products andintermediates. There is also a need for an instrument for manipulatingthe microsystem platform to effect fluid movement, thermal control,reagent mixing, reactant detection, data acquisition, data analysis anddata and systems interface with a user. Such devices are needed, inalternative embodiments, that are sophisticated (for professional, e.g.,hospital, use), easy to use (for consumer, e.g., at-home monitoring,uses) and portable (for field, e.g., environmental testing, use). Suchdevices also advantageously combine “wet” chemistry capabilities withinformation processing, storing and manipulating ability.

SUMMARY OF THE INVENTION

This invention provides an integrated, microanalytical/microsyntheticsystem for performing a wide variety of biological, biochemical andchemical analyses on a microminiature scale. The invention providesapparatus and methods for performing such microscale processes on amicroplatform, whereby fluid is moved on the platform in definedchannels motivated by centripetal force arising from rotation of theplatform.

In one aspect of the invention is provided amicroanalytic/microsynthetic system comprising a combination of twoelements. The first element is a microplatform that is a rotatablestructure, most preferably a disk, the disk comprising sample, inletports, fluid microchannels, reagent reservoirs, reaction chambers,detection chambers and sample outlet ports. The disk is rotated atspeeds from about 1-30,000 rpm for generating centripetal accelerationthat enables fluid movement. The disks of the invention also preferablycomprise fluid inlet ports, air outlet ports and air displacementchannels. The fluid inlet ports allow samples to enter the disk forprocessing and/or analysis. The air outlet ports and in particular theair displacement ports provide a means for fluids to displace air, thusensuring uninhibited movement of fluids on the disk. Specific sites onthe disk also preferably comprise elements that allow fluids to beanalyzed, including thermal sources, light, particularly monochromaticlight, sources, and acoustic sources, as well as detectors for each ofthese effectors. Alternatively, some or all of these elements can becontained on a second disk that is placed in optical or direct physicalcontact with the first.

The second element of the invention is a micromanipulation device thatis a disk player/reader device that controls the function of the disk.This device comprises mechanisms and motors that enable the disk to beloaded and spun. In addition, the device provides means for a user tooperate the Microsystems in the disk and access and analyze data,preferably using a keypad and computer display.

The invention provides methods and apparatus for the manipulation ofsamples consisting of fluids, cells and/or particles containing orcomprising an analyte. The microplatform disks of the invention compriseMicrosystems composed of, but no restricted to, sample input ports,microchannels, chambers, valves, heaters, chillers, electrophoretic anddetection systems upon a disk. Movement of the sample is facilitated bythe judicious incorporation of air holes and air displacement channelsthat allow air to be displaced but prevent fluid and/or particle lossupon acceleration.

A preferred embodiment of the disk of the invention incorporatesmicromachined mechanical, optical, and fluidic control structures (or“systems”) on a substrate that is preferably made from plastic, silica,quartz, metal or ceramic. These structures are constructed on asub-millimeter scale by photolithography, etching, stamping or otherappropriate means.

Sample movement is controlled by centripetal or linear acceleration andthe selective activation of valves on the disk.

In preferred embodiments of the invention, a section of the disk isdedicated to information processing by standard read/write digitaltechnology. Data resulting from processing and analysis is recorded onthe disk surface using digital recording means. In additional preferredembodiments, read-only memory (ROM) on the disk comprises diskinformation, instructions, experimental protocols, data analysis andstatistical methods that can be accessed by a user operating the disk.

The process of fluid transport by centripetal acceleration and themicromanipulation device that enables such processing have a-wide rangeof applications in the synthesis and analysis of fluids and detection ofanalytes comprising a fluid, particularly a biological fluid. Chemicaland biochemical reactions are performed in a reaction chamber on thedisk by the selective opening of contiguous reagent chambers by means ofcapillary, mechanical or thermal valve mechanisms. The contents of thosechambers are delivered to the reaction chamber with the application ofcentripetal acceleration. The product of the reaction can then be usedas a reagent for subsequent reactions, interrogated by detection systemsor recovered.

Certain preferred embodiments of the apparatus of the invention aredescribed in greater detail in the following sections of thisapplication and in the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (top view) and 1B (side view) illustrate the arrangement ofreservoirs (12,14,18,20), valves (13,15,17,19,21,23,25) reactionchambers (16,22,24), ports (11,32) and air vents (29,33,34,35) in diskscomprising the microplatforms of the invention. FIG. 1C shows thearrangement of a multiplicity of Microsystems on a disk.

FIG. 2A is a graph and FIG. 2B is a schematic diagram of the arrangementof a channel on a disk of the invention as described with relation toEquation 5.

FIG. 3A is a graph and FIG. 3B is a schematic diagram of the arrangementof a channel on a disk of the invention as described with relation toEquations 12 and 13.

FIG. 4A is a graph and FIG. 4B is a schematic diagram of the arrangementof a channel on a disk of the invention as described with relation toEquation 14.

FIGS. 5A, 5B and 5C are graphs and FIG. 5D is a schematic diagram of thearrangement of a channel on a disk of the invention as described withrelation to Equation 15.

FIG. 6 is a schematic diagram of a piezoelectric stack microvalve.

FIG. 7 is a schematic diagram of a pneumatically-activated microvalve.

FIG. 8 is a schematic diagram of device to deliver pneumatic pressure toa revolving disk.

FIG. 9 is a schematic diagram of a bimetallic microvalve.

FIG. 10 is a schematic diagram of a pressure-balanced microvalve.

FIG. 11 is a schematic diagram of a polymeric relaxation microvalve.

FIGS. 12A and 12B represent two different embodiments of fluorescencedetectors of the invention.

FIGS. 13A, 13B and 13C are a schematic diagrams of a multiple loadingdevice for the disk.

FIGS. 14A through 14F illustrate laser light-activated CD-ROM capabilityof the disk of the invention.

FIG. 15 is a flow diagram of the processor control structure of aplayer/reader device of the invention.

FIG. 16 is a schematic diagram of a transverse spectroscopic detectionchamber.

FIGS. 17A through 17E are schematic diagrams of the different structuraland functional layers of a disk of the invention configured for DNAsequencing.

FIG. 17F is a schematic diagram of basic zones and design formats foranalytic disks.

FIG. 17G is a schematic diagram of a disk configured as a home testdiagnostic disk.

FIG. 17H is a schematic diagram of a disk configured as a simplifiedimmunocapacitance assay.

FIG. 17I is a schematic diagram of a disk configured as a gas andparticle disk.

FIG. 17J is a schematic diagram of a hybrid disk comprisingseparately-assembled chips.

FIG. 17K is a schematic diagram of a sample authorizing disk.

FIG. 17L is a schematic diagram of a disk configured for pathologicalapplications.

FIG. 17M is a schematic diagram of a disk with removable assay layers.

FIG. 17N is a schematic diagram of a disk for assaying aerosols.

FIG. 170 is a schematic diagram of a disk for flow cytometry.

FIG. 17P is a schematic diagram of a disk for microscopy applications.

FIG. 17Q is a schematic diagram of a disk for immunoassay applications.

FIG. 17R is a schematic diagram of a thin-layer chromatography disk.

FIG. 18 is a schematic diagram of a disk configured for hematocritdetermination.

FIG. 19 is a schematic diagram of a disk configured for SPLITTfractionation of blood components.

FIG. 20 is a schematic diagram of a disk configured as a DNA meltometer.

FIG. 21 is a schematic diagram of a disk configured for DNAamplification.

FIG. 22 is a schematic diagram of a disk configured for automatedrestriction enzyme digestion of DNA.

FIG. 23 is a schematic diagram of a portion of a disk microsystemconfigured for DNA synthesis.

FIG. 23B is a schematic diagram of a disk configured for a multiplicityof DNA oligonucleotide syntheses.

FIG. 24 is a schematic diagram of a disk configured for DNA sequencing.

FIG. 25 is a schematic diagram of a disk configured for iron assay.

FIG. 26 is a schematic diagram of a disk configured for solid phasereaction.

FIG. 27 is a schematic diagram of a disk configured for sampleextraction.

FIG. 28 is a schematic diagram of a disk configured for gel or capillaryelectrophoresis.

FIG. 29 is a schematic diagram of a transverse optical path in amicroplatform.

FIG. 30 is a block diagram of process flow in controlling informatics ofthe invention.

FIGS. 31A and 31B are a more detailed schematic diagram of controllinginformatics of the invention.

FIGS. 32A and 32B are a more detailed schematic diagram of controllinginformatics of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention provides a microplatform and a micromanipulation devicefor performing microanalytical and microsynthetic assays of biological,chemical, environmental and industrial samples. For the purposes of thisinvention, the term “sample” will be understood to encompass anychemical or particulate species of interest, either isolated or detectedas a constituent of a more complex mixture, or synthesized fromprecursor species. The invention provides a combination of amicroplatform that is a rotatable, analytic/synthetic microvolume assayplatform (collectively referred to herein as a “disk”) and amicromanipulation device for manipulating the platform to achieve fluidmovement on the platform arising from centripetal force on the platformas result of rotation. The platform of the invention is preferably andadvantageously a circular disk; however, any platform capable of beingrotated to impart centripetal for a fluid on the platform is intended tofall within the scope of the invention.

The microplatforms of the invention (preferably and hereinaftercollectively referred to as “disks”; for the purposes of this invention,the terms “microplatform”, “microsystems platform” and “disk” areconsidered to be interchangeable), are provided to comprise one or amultiplicity of microsynthetic or microanalytic systems. Suchmicrosynthetic or microanalytic systems in turn comprise combinations ofrelated components as described in further detail herein that areoperably interconnected to allow fluid flow between components uponrotation of the disk. These components can be fabricated as describedbelow either integral to the disk or as modules attached to, placedupon, in contact with or embedded in the disk. The invention alsocomprises a micromanipulation device for manipulating the disks of theinvention, wherein the disk is rotated within the device to providecentripetal force to effect fluid flow on the disk. Accordingly, thedevice provides means for rotating the disk at a controlled rotationalvelocity, for stopping and starting disk rotation, and advantageouslyfor changing the direction of rotation of the disk. Bothelectromechanical means and control means, as further described herein,are provided as components of the devices of the invention. Userinterface means (such as a keypad and a display) are also provided.

The invention provides methods and apparatus for the manipulation ofsamples consisting of fluids, cells and/or particles (generically termed“sample” herein) containing an analyte of interest. The platforms of theinvention consist of systems comprising sample input ports,microchannels for fluid flow, reagent reservoirs, mixing chambers,reaction chambers, optical reading chambers, valves for controllingfluid flow between components, temperature control elements, separationchannels, electrophoresis channels and electrodes, air outlet ports,sample outlet ports, product outlet ports, mixing means includingmagnetic, acoustic and mechanical mixers, an on-board power supply suchas a battery or electromagnetic generator, liquid and dry reagents, andother components as described herein or known to the skilled artisan.The movement of the sample is facilitated by the judicious incorporationof air holes or air displacement channels that allow air to be displacedbut prevent fluid and/or particle loss upon acceleration. Preferably,the disk incorporates microfabricated mechanical, optical, and fluidiccontrol components on platforms made from, for example, plastic, silica,quartz, metal or ceramic. For the purposes of this invention, the term“microfabricated” refers to processes that allow production of thesestructures on the sub-millimeter scale. These processes include but arenot restricted to photolithography, etching, stamping and other meansthat are familiar to those skilled in the art.

Fluid (including reagents, samples and other liquid components) movementis controlled by centripetal acceleration due to rotation of theplatform, and by the selective activation of valves controlling theconnections between the components of the Microsystems of the platform.The magnitude of centripetal acceleration required for fluid to flow ata rate and under a pressure appropriate for a particular microsystem isdetermined by factors including but not limited to the effective radiusof the platform, the position angle of the structures on the platformwith respect to the direction of rotation and the speed of rotation ofthe platform.

Chemical and biochemical reactions are performed in a reaction chamberby the selective opening of microvalves controlling access to contiguousreagent reservoirs. Microvalves as described in more detail belowinclude mechanical, electrical and thermal valve mechanisms, as well ascapillary microvalves wherein fluid flow is controlled by therelationship between capillary forces and centripetal forces acting onthe fluid. The contents of the reagent reservoirs, that are connected areaction chamber through microchannels controlled by such microvalves,are delivered to the reaction chamber by the coincident rotation of themicroplatform and opening of the appropriate microvalves. The amount ofreagent delivered to a reaction chamber is controlled by the speed ofrotation and the time during which the valve to the reagent reservoirsis open. Products of the reaction performed in the reaction chamber aresimilarly removed from the reaction chamber to an analytical array, asecond reaction chamber or a product outlet port by the controlledopening of microvalves in the reaction chamber.

Analytical arrays constituting components of the microplatforms of theinvention include detection systems for detecting, monitoring,quantitating or analyzing reaction course, products or side-products.Detection systems useful in the fabrication and use of themicroplatforms of the invention include, but are not limited to,fluorescent, chemiluminescent, colorimetric, electrochemical andradioactivity detecting means. Optionally, the detection system can beintegral to the platform, comprise a component of the devicemanipulating the platform, or both.

Thus, the microplatform and micromanipulation device provided by theinvention produce analytic or synthetic data to be processed. Dataprocessing is accomplished either by a processor and memory module onthe disk, by the device microprocessor and memory, or by an out boardcomputer connected to the micromanipulation device. Removable media fordata retrieval and storage is provided either by the disk itself or bythe device, using computer diskette, tape, or optical media.Alternatively and advantageously, data is written on the microplatformusing CD read/write technologies and conventional optical data storagesystems In such embodiments, data is written to the microplatform on theunderside of the platform, opposite to the “wet” chemistry side holdingthe various microsystem components disclosed herein.

The physical parameters of the microplatforms of the invention arewidely variable. When provided as a disk, the disk radius ranges from1-25 cm, and disk thickness ranges from 0.1 mm to 10 cm, more preferably0.1 mm to 100 mm. Preferred embodiments that are most advantageous formanufacturing and operation of the disks of the invention havedimensions within one or more of four pre-existing formats: (1) 3-inchcompact disk (CD), having a radius of about 3.8 cm and thickness ofabout 1 mm: (2) 5-inch CD, having a radius of about 6 cm and aqathickness of 1 mm; (3) 8-inch CDV (commercially termed a “Laservision”disk), having a radius of 10 crm and a thickness of 2 mm; and (4)12-inch CDV disk, having a radius of 15 cm and a thickness of 2 mm.

Microchannel and reservoir sizes are optimally determined by specificapplications and by the amount of reagent and reagent delivery ratesrequired for each particular embodiment of the microanalytic and microsynthetic methods of the invention. For microanalytical applications,for example, disk dimensions of a 5-in CD (6 cm×1 mm) are preferred,allowing reagent reservoirs to contain up to 0.5 mL (close to the actualdisplaced by the disk). Microchannel sizes can range from 0.1 μm to avalue close to the 1 mm thickness of the disk. Microchannel andreservoir shapes can be trapezoid, circular or other geometric shapes asrequired; Microchannels preferably are embedded in a microsystemplatform having a thickness of about 0.1 μm to 100 mm, wherein thecross-sectional dimension of the microchannels across the thicknessdimension of the platform is less than 500 μm and from 1 to 90 percentof said cross-sectional dimension of the platform. Reagent reservoirs,reaction chambers, detections chambers and sample inlet and outlet portspreferably are embedded in a microsystem platform having a thickness ofabout 0.1 μm to 100 mm, wherein the cross-sectional dimension of themicrochannels across the thickness direction of the platform is from 1to 75 percent of said cross-sectional dimension of the platform;

Input and output (entry and exit) ports are components of themicroplatforms of the invention that are used for the introduction ofremoval of a variety of fluid components. Entry ports are provided toallow samples and reagents to be placed on or injected onto the disk;these types of ports are generally located towards the center of thedisk. Exit ports are provided to allow air to escape, advantageouslyinto an on-disk “muffler” or “baffle” system, to enable uninhibitedfluid movement on the disk. Also included in air handling systems on thedisk are air displacement channels, whereby the movement of fluidsdisplaces air through channels that connect to the fluid-containingmicrochannels retrograde to the direction of movement of the fluid,thereby providing a positive pressure to further motivate movement ofthe fluid. Exit ports are also provided to allow products to be removedfrom the disk. Port shape and design vary according specificapplications. For example, sample input ports are designed, inter alia,to allow capillary action to efficiently draw the sample into the disk.In addition, ports can be configured to enable automated sample/reagentloading or product removal. Entry and exit ports are most advantageouslyprovided in arrays, whereby multiple samples are applied to the diskusing a specifically-designed administration tool. Similar tools areuseful designed to effect product removal from the microplatform.Representative arrangements of sample ports, air vents, reagentreservoirs, reaction chambers and microvalves are shown in FIGS. 1Athrough 1C.

Operative and optimal placement of the various disk components andelements depend on the dynamics of fluid movement in relation tocentripetal forces. Centripetal force is a function of platform radius,disk rotation speed and fluid density. Certain functional parametersrelevant to the platform microsystems of this invention are understoodin terms of the following equations. These should represent limits ofsystem performance, because they assume both viscous and non-viscous(turbulent) losses for fully-developed fluid flow.

The driving force for fluid motion or creating fluid pressures is theforce on matter which results from centripetal acceleration. A devicemay rotate at an angular rate of f in revolutions/sec and angularfrequencyω=2πf  (1)The centripetal acceleration (or acceleration oriented along the radiusat a radial distance R from the center of the uniformly-rotating disk)isa_(c)=ω²R  (2)A mass m in such uniform circular motion is subject to a centripetalforceF_(c)=ma_(c)=mω²R  (3)which is directed inward along the radius to the center of rotation. Ifthe mass is held fixed at this radius, the device causing rotationsupplies this force; this is the origin of the static pressure in liquidcolumns discussed below. If the mass is placed on top of a trap-doorabove a radially-oriented tube, and the trap-door opened, the inertia ofthe mass will cause it to accelerate down the tube; this is the basisfor driving fluids radially outward on a rotating disk.

Rotation may create a static pressure in a non-flowing fluid. Assume acolumn of liquid extending from an inner radius R₀. The tube may bealong the radius or inclined at an angle to the radius. Let the pressureat position R₀ be defined as P₀, which is for example atmosphericpressure. The excess pressure due to rotation of the liquid at PositionR such that R₀<R is found by integrating the centripetal force per unitarea for liquid of density p from position R₀ to R:P−P ₀=∫ρa_(c)=ρω²/2×(R ² −R ₀ ²)  (4)If the tube is filled, extending from the center, then this pressure isP−P ₀=(2.834×10⁻⁴)pf ² R ²  (5)in pounds per square inch (psi) where R=radial position in cm, ρ=densityin gm/cm³, and f=frequency in revolutions/sec. Thus, the pressure (orthe amount of centripetal force on a fluid) varies directly with thedensity of the fluid, and as the square of the radial position from thecenter of rotation as well as the square of the frequency of rotation.

To determine the velocity of liquid in motion in channels on a rotatingdisk, the equation of motion for the fluid must be solved. An element offluid of radius a and length dR filling the circular channel has a massdm subject to acceleration:dm=πρa²dR  (6)

The equation of motion for this fluid element isforce=(mass)×(acceleration). The forces are centripetal forces,capillary forces due to differences in interfacial energies between thefluid and vapor and fluid and solid surfaces, and dissipative forces dueto the viscosity of the liquid and nonuniformity of flow. Capillaryforces are ignored; it is understood that centripetal force and/orexternal pressure may need to be applied to force liquid into channelswhich are not wetted. As an over-estimate of these dissipative forces,both the force for fully-developed laminar flow of a Newtonian fluid(F_(L)) and that due to non-uniform flow (F_(D)) are included:F=maF _(c) +F _(L) +F _(D) =dma _(R)F _(c) +F _(L) +F _(D)=(ρπa ² dR)a _(R)  (7)where a_(R) is the acceleration of the fluid mass element along theradius andF _(c)=(ρπa² dR)ω² RF _(L)=−(8μπa ² dR)uF _(D)=−(2ρπa ² dR)u ²  (8)where μ is the viscosity and u is the radial velocity of the fluid.These last two expressions are standard-mechanics expressions forfully-developed and completely undeveloped laminar flow, such as atchannel entrances/exits or at the ends of a flowing droplet. Also notethat for tubes or channels inclined at an angle θ with respect to theradius F_(c) would be replaced by (F_(c))×cos θ. The final equationbecomes(ρπa ² dR)ω² R−(8μπdR)u−(2ρπa ² u ² dR)=(ρπra ² dR)(du/dt)  (9)where the radial acceleration of the fluid is defined by a_(R)−(du/dt).This is a differential equation for the fluid flow velocity.

This equation is solved for specific examples. Consider a droplet offluid of length L moving in a radial channel of greater length than thedroplet.

Because the fluid in the droplet all moves at the same velocity, dR maybe replaced by L and R by the average position of the droplet,<R>=(R+L/2).

Dividing out common factors:(ω² (R+L/2)/2)−(8 μ/ρa ²)u−2(u ² /L)=(du/dt)  (10)This equation must be solved numerically. An approximation may be madewhich has been justified through comparison with numerical solutions. Itconsists of this: the negative terms on the left-hand-side almostentirely cancel the positive term. Then the right-hand-side can be setto 0 and a solution can be made to the resultant equation for the“terminal velocity” at position R, u₀(ω² (R+L/2)/2)−(8μ/ρa ²)u ₀−₂(u ₀ ² /L)=0  (11)This is a quadratic equation which has the solutionu ₀=−(B+{square root}{square root over ( )}B ²+4AC)/2A  (12)withA=L/2B=8μ/ρa ²C=(ω² (R+L/2)/2)  (13)In conventional units these become A=2/L, B=320μ/ρD² andC=(19.74)f²(2R+L) with u₀=fluid velocity in cm/sec; L=droplet length incm; μ=viscosity in poise; p=fluid density in gm/cm³; D=2a=tube diameterin cm; and R=radial position of the fluid droplet in cm. As described,this expression gives the approximate velocity of a droplet of fluid ina tubular channel, the volume of the droplet resulting in droplet lengthbeing shorter than the channel length. This estimate assumes bothviscous and non-viscous losses. The velocity of a fluid droplet willincrease with increasing density and droplet volume (length), anddecrease with increased viscosity. The velocity will increase withincreased channel diameter, rotational velocity, and radial position.

Fluid flow velocity in a filled channel connecting a full chamber atposition R₀ and receiving reservoir at position R₁ is calculated bydefining L in equation (11) and subsequent equations as the channellength, L=R, −R. Then equation (13) with the definitions followingequation (13) are used to calculate the flow velocity in the filledchamber as a function of radius.

The rate of fluid-flow is the product of velocity and channel area:Q=u ₀ πa ² =u ₀ πD ²/4  (14)where Q=flow in mL/sec; u₀=velocity in cm/sec (calculated from equations12 and 13); and D=tube diameter in cm.

The time required to transfer a volume V from a reservoir to areceptacle through a tube or channel of length L depends on whether V issuch that the tube is filled (length of a “droplet” of volume V in thetube would be longer than the tube itself) or unfilled by volume V. Inthe former case, this time is approximately the volume V of the fluiddivided by the rate of flow Q; in the latter case it is approximatelythis calculated time multiplied by the ratio of the tube length to theresultant droplet length:Dt=V/Q, if L≦(4V/πD2)Dt=(V/Q)×(4πD ² L/4V), if L>(4V/πD ²)  (15)wherein D_(t) is the same time in seconds for fluid of volume V in mLflowing at rate Q in mL/sec to flow from a filled reservoir to areceptacle through a tube of length L and diameter D in cm. The rate offlow Q is calculated from eq. (14) and by extension equations (12) and(13) and the definitions of the parameters following equation (13). Thetime Dt increases with increasing volume transferred and decreases withincreasing flow-rate.

Fluid characteristics such as pressure and velocity are related tophysical parameters of the disk, such as disk radius and speed ofrotation, as described above. These relationships are illustrated inFIGS. 2-5, derived from the above equations for water at roomtemperature, with p=1 gm/cm³ and μ=0.001 poise. These figures delineatethe most relevant parameters of fluid movement on a rotating disk.

FIG. 2A illustrates the relationship between static pressure in afluid-filled tube 30 cm in length as a function of radial distance®) androtation rate (f), calculated from Equation 5. The arrangement of thetube on a rotating disk is shown in FIG. 2B. It can be seen thatpressures of between 0 and 10,000 psi can be generated in the tube atrotational speeds of 0 to 10,000 rpm. Pressures of this magnitude areconventionally used, for example, to drive high pressure liquidchromatography (HPLC).

FIG. 3A shows the radial velocity of droplets having volume of 1, 10 and100 μL droplets moving in an empty, 30 cm long tube with a diameter of 1mm, calculated from Equations 12 and 13. The rube is aligned to extendalong the radius of the disk from the center, and the disk is rotated atspeeds of 100, 1,000 or 10,000 rpm. The arrangement of the tube on arotating disk is shown in FIG. 3B. These velocities may be used tocalculate the transfer time for fluid droplets. For example, a 1 μLdroplet flows at approximately 20 cm/sec when at a position 2 cm fromthe center of a disk rotating at 1,000 rpm. Hence, the time to flowthrough a 1 cm tube can be calculated to be about 0.05 seconds. (Fortubes oriented non-radially at an angle of 45° to the direction ofrotation, the velocity drops by a factor of 30%.)

FIG. 4A illustrates flow rates in a 5 cm fluid-filled tube of differentdiameters. The tubes are each placed on a rotating disk and connects tworadially oriented reservoirs, shown in FIG. 4B. According to Equation14, flow rates are a function of radial position of the tube (which varyin this example from 2-30 cm), the tube diameter (10 μm, 100 μm, or 1000μm), and rotation frequency (100, 1,000 or 10,000 rpm). (As above, fortubes with a non-radial orientation of 45°, the velocity drops by afactor of 30%). Droplet velocities shown in FIG. 3A were calculated byEquation 3 and flow rates determined using Equation 4.

In FIGS. 5A, 5B and 5C, the time required to transfer 1, 10, and 100 μLdroplets, respectively, through a 5 cm tube is shown. The tube connectstwo radially oriented reservoirs as illustrated in FIG. 5D. Transfertimes are a function of radial position of the tube (o-30 cm), tubediameter (10 μm, 100 μm, or 1,000 μm), and rotation frequency (100,1,000 or 10,000 rpm). The curves shown in FIGS. 5A, 5B and 5C werecalculated using Equation 15.

Taken together, these formulae and graphs describe the interrelationshipof disk radii and rotation speeds, channel lengths and diameters, andfluid properties such as viscosity and density in determining fluidvelocities and flow rates on the disk. The assumptions behind thesederivations include viscous losses due to Poiseuille (non-turbulent)flow with the addition of losses due to non-uniform flow of droplets andat tube inlet and outlet ports. These formulae and graphs provide lowerlimits for velocities and flow rates. Fluid velocities can range fromless than 1 cm/sec to more than 1,000 cm/sec, and fluid flow rates fromless than 1 pL/sec to tens of mL/sec for rotation rates ranging from 1to 30,000 rpm. By combining channel diameters and positions on the disk,it is possible to carry out fluid transfer over a wide range of timescales, from milliseconds to hours and tens of hours for variousprocesses.

Disk Coatings and Composition

Microplatforms such as disks and the components comprising suchplatforms are advantageously provided having a variety of compositionand surface coatings appropriate for a particular application among thewide range of applications disclosed herein. Disk composition will be afunction of structural requirements, manufacturing processes, andreagent compatibility/chemical resistance properties. Specifically,disks are provided that are made from inorganic crystalline or amorphousmaterials, e.g. silicon, silica, quartz, metals, or from organicmaterials such as plastics, for example, poly(methyl methacrylate)(PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate,polyethylene, polystyrene, polyolefins, polypropylene and metallocene.These may be used with unmodified or modified surfaces as describedbelow.

One important structural consideration in the fabrication of theMicrosystems disks of the invention is mechanical failure due to stressduring use. Failure mechanisms for disks rotated at high velocitiesinclude fracture, which can arise as the result of tensile loading, ordue to cracking and crazing, as described on Hertzberg (1989,Deformation and Fracture Mechanics of Engineering Materials, 3rdedition, Wiley & Sons: New York). These failures occur when the stress(defined as the load per unit area) due to rotation of the disk exceedsa critical value characteristic of the material used to make the disk.The “load” at any point in the disk is the force of tension due torotation; i.e., at a given radius on the disk, the overall load is thecentripetal force necessary to keep the material at larger radii movingcircularly; the load/area or stress is then this force divided by thetotal area of the disk (2πr×the thickness of the disk). The criticalvalue of stress at which a material will fail is termed the yieldstress, and it depends on the cohesive energy binding the materialtogether and the presence of defects in the material (such ascrystalline defects in silicon or plastic substrate material). Adefect-free material can be torn apart, whereas small defects willpropagate through cracking or “crazing” (i.e., plastic deformation andfailure of a formerly glassy plastic). For example, the yield strengthof commercial silicon permits a 30 cm disk to be spun at 10,000 rpmwithout mechanical failure when the diameter of internal channels andchambers is less than approximately 80% of the total thickness of thedisk. In disks made of plastics, stresses on the disk are reduced ingeneral due to the lower density of the plastic (which reduces theload/unit area). However, the yield strengths are also smaller by abouttwo orders of magnitude than in silicon (as described in greater detailin Luis & Yannis, 1992, Computational Modeling of Polymers, (Bureitz,ed.), Marcel Dekker: New York). One solution to this problem is providedeither by spinning a plastic 30 cm disk at a slower speed (such as 1,000rpm), or increasing the size of the disk radius (such as using a 4 cmplastic disk for applications requiring 10,000 rpm rotation speeds).Thus, material choice specific for a particular application issufficient to accommodate disk composition-related constraints on diskfunctional properties and characteristics.

Disk material in contact with fluids must also be resistant todegradation by reagent solutions (such as acetonitrile, polyacrylamide,high- or low-pH buffers) under rotational stress, upon heating andcooling, and in response to illumination with high-intensity ultravioletor visible light (occurring, inter alia, with the use of certaindetection means as described below). In addition, the surfaces presentedto reagents and reaction mixtures (such as microchannels, reservoirs andreaction chambers) must have desirable surface properties appropriatefor each application. Silicon, silica, and quartz are especially robustmaterials as substrates for microplatform fabrication. Silicon and itsoxides (essentially silica) are chemically attacked only by someperoxides (such as a mixture of hydrogen peroxide plus sulfuric acid),hydroxides (such as KOH), hydrofluoric acid (HF), either alone or incombination with alkali-based nitrates, and various perfluorinatedsolvents (like SF₆) see Iler, 1979, The Chemistry of Silica, Wiley &Sons: New York; Properties of Silicon, Xth ed., INSPEC:, London, 1988).Silicon-based substrates are chemically inert to aliphatic and aromatichydrocarbons (such as tetrahydrofuran, toluene, and the like), and aresubstantially inert when exposed to water and neutral aqueous solutions.

A wide variety of polymer-based (plastics) substrates are suitable forfabricating Microsystems platforms of the invention. The mostchemically-resistant polymer, poly(tetrafluoroethylene; PTFE), is notmelt-processible but may be easily machined. PTFE is virtuallychemically inert and can be used in most applications utilizing strongacids, bases, alkalis, halogenated solvents, or other strong chemicalreagents. Other fluoropolymers (such as FEP, PFA) are more easilyprocessed than PTFE and retain most of PTFE's chemical resistance. Moreeasily-processed materials may be chosen for selective resistance: forexample, although polyimides are highly resistant to alcohols, alkalis,aliphatic hydrocarbons, and bases (e.g., NaOH), their resistance topartially-halogenated solvents (e.g. dichlorobenzene) is poor. Poly(vinyl chloride) is strongly resistant to oxidizing acids and aliphatichydrocarbons, while its resistance to aromatic compounds is poor. Inaddition, many materials that are not highly-resistant to concentratedapplications of certain chemicals provide sufficient resistance todilute solutions or provide sufficient resistance for single-use devices(e.g., polyamides and polyimides may be used with dilute solutions ofcertain acids such as acetic acid and hydrochloric acid). Most polymericmaterials are resistant to water.

Specific chemical/polymer combinations include: formamide, lutidine, andacetonitrile with non-aromatic, non-polar polymers (polypropylene,polyethylene); dichloromethane with polycarbonates and aromatic polymers(polystyrene); ethanolamine and dimethyl sulfoxide with aliphatic andnon-aromatic polymers (poly(methyl methacrylates), polyimides,polyamides). Fluoropolymers are resistant to all of the above chemicalagents. Other solvents and reagents of interest, including pyridine,tetrazole, trichloracetic acid, iodine, acetic anhydride,-methylpyrrolidine, N,N-diethylpropylethylamine and piperidine, aresuitable for use with fluoropolymers and some solvent resistantpolymers, such as PVC (Encyclopedia of Polymer Science and Technology,2^(nd) ed., v. 3, pp 421-430, X ed., John Wiley & Sons, New York, 1989).A small set of such materials provides sufficient flexibility forvirtually any application.

The surface properties of these materials may be modified for specificapplications. For example, appropriate surface-modification can eitherencourage or suppress cell and/or protein absorption. Surfacemodification can be achieved by silanization, ion implantation andchemical treatment with inert-gas plasmas (i.e., gases through whichelectrical currents are passed to create ionization). A strongcorrelation has been established between water contact angle and celladsorption, with hydrophilic surfaces showing significantly less celladsorption than hydrophobic surfaces (see Ikada, 1994, Biomaterials 15:725). Silicon, silica, and quartz present and inherently high-energy,hydrophilic surface. Alteration of surface properties is attainedthrough hydroxylation (achieved by NaOH treatment at high temperatures)or silanization. Silanes and siloxanes are particularly appropriate forincreasing the hydrophilicity of an otherwise hydrophobic surface. Thesecompounds consist of one or several reactive head-groups which bond(chemically or through hydrogen-bonding) to a substrate, for example, acore region of alkane (—CH₂O—). These compounds also provide a route formore sophisticated alteration of surface properties (such as derivationwith functional groups to obtain the surface properties of interest). Awide variety of such functionalities can be introduced at a surface,including vinyl, phenyl, methylene and methoxy groups, as well assurfaces providing mixed functionalities. These functional groups notonly change gross properties like liquid contact angle, but providesites for preferential adsorption of molecules, either per se or as aresult of further conjugation of specific binding moieties such aspeptides, antibodies or the like. Silation is most often accomplishedthrough immersion in aqueous solution at slightly-elevated temperatures.The chemical resistance of silane and siloxane coatings is determined bythe nature of bonding within the chemisorbed molecule (Arkles, 1977,Chemtech 7: 125). It should be noted that such properties ashydrophobicity are maintained for significant periods when organosilanesare in contact with quite corrosive acids, implying that single-use orlimited-use applications in these environments are possible.

Plastic-based disk can also be readily treated to achieve the requiredsurface properties. Inert-gas or reactive-gas plasmas are commonly usedto alter surface energies through the formation of surface complexes,for example, hydroxyl-rich surfaces for increased hydrophilicity, orperfluorinated surfaces for increased hydrophobicity. Surface graftpolymerization is a technique used to graft polymers or oligomers withthe desired surface properties to a substrate polymer chosen for itsbulk processability and manufacturing properties, such as a plastic.Commercial methods for initiating graft polymerization include gammaradiation, laser radiation, thermal or mechanical processing,photochemical processes, plasma, and wet chemical processes (furtherdiscussed in Encyclopedia of PolymerScienceand Technology, 2^(nd) ed.,(Supplement), Wiley & Sons: New York, 1989, pp 675-689). Chemicalmodification of polymer surfaces (and appropriate polymers) includesoxidations (polyethylenes), reductions (fluoropolymers), sulfonations,dehydrohalogenations (dehydrofluorination of poly (vinylidene fluoride),and hydrolyses. While the chemical nature of the surface is alteredthrough chemical modification, mechanical properties, durability andchemical resistance are primarily a function of the substrate plastic.For example, surface grafting of poly(ethylene glycol) (PEG) ontopolyethylene yields a: surface that is both hydrophilic (unlikepolyethylene) and resistant to water (PEG is itself soluble in water,while polyethylene is not). Finally, silation of organic polymersurfaces can also be performed, providing a wide variety of surfaceenergy/chemistry combinations.

Embodiments comprising thin film disks are provided, comprising “layers”of Microsystems disks stacked on a solid support, are useful forsequential assay with conservation of the disk and efficient andinexpensive use of the microsystem-comprising disks as consumables. Anillustration of such disks are shown in FIG. 17L. Such disks are capableof being uniquely identified, for example, by imprinting a barcodedirectly on the disk.

Particular examples of disks fabricated for a variety of applications isprovided below in the Examples.

Disk-Related Devices and Elements

Microsystems platforms (microplatforms) of the invention are providedwith a multiplicity of on-board components, either fabricated directlyonto the disk, or placed on the disk as prefabricated modules. Inaddition to be integral components of the disk, certain devices andelements can be located external to the disk, optimally positioned on adevice of the invention, or placed in contact with the disk.

1. Temperature Control Elements

Temperature control elements, particularly heating elements, includeheat lamps, direct laser heaters, Peltier heat pumps, resistive heaters,ultrasonication heaters and microwave excitation heaters. Coolingelements include Peltier devices and heat sinks, radiative heat fins andother components to facilitate radiative heat loss. Thermal devices canbe applied to the disk as a whole or in specific areas on the disk. Thethermal elements can be fabricated directly onto the disk, or can befabricated independently and integrated onto the disk. Thermal elementscan also be positioned external to the disk. The temperature of anyparticular area on the disk is monitored by resistive temperaturedevices (RTD), thermistors, liquid crystal birefringence sensors or byinfrared interrogation using IR-specific detectors. Temperature at anyparticular region of the disk can be regulated by feedback controlsystems. A micro-scale thermo-control system can be fabricated directlyon the disk, fabricated on a microchip and integrated into the disk orcontrolled through a system positioned external to the disk.

2. Filters

Filters, sieving structures and other means for selectively retaining orfacilitating passage of particulate matter, including cells, cellaggregates, protein aggregates, or other particulate matter comprisingfluids applied to a microanalytical or microsynthetic disk of theinvention. Such filtering means include microsieving structures that arefabricated directly into a fluid handling structure on the disk (e.g.,U.S. Pat. No. 5,304,487; International Application, Publication No.WO93/22053; Wilding et al., 1994, Automat. Analyt. Tech. 40: 43-47) orfabricated separately and assembled into the fluid handling structures.The sieving structures are provided with a range of size exclusionorifices and are optionally arranged sequentially so as to fractionate asample based upon the sizes of the constituent parts of the sample.

Other types of filters include materials that selectively remove sampleconstituents based on electrostatic forces between the filter materialand the sample constituents. The electrostatic composition of thesieving materials may be inherent to the material or bestowed upon it byvirtue of a charge delivered to the material through an electroniccircuit. The materials captured by the filter material can beirreversibly bound or can be selectively eluted for further processingby adjusting the composition and ionic strength of buffers or, in thecase of an electronically regulated material, by modulating theelectronic state of the material.

In yet other embodiments of the filters of the microsystem platforms ofthis invention, specific components of a sample can be retained in asection, microchannel or reservoir of a disk of the invention byinteraction with specific proteins, peptides, antibodies or fragmentsthereof derivatized to be retained within the surface of a component ofthe disk. Materials captured by such specific binding can be eluted fromthe surface of the disk and transferred to a collection reservoir bytreatment with appropriately-chosen ionic strength buffers, usingconventional methods developed for immunological or chromatographictechniques.

The invention also provides compartments defined by sections of amicrochannel or by a chamber or reservoir wherein the inlet and outletports of the chamber are delimited by a filtering apparatus. In certainembodiments, the chamber thus defined contains a reagent such as a beadand particularly a bead coated with a compound such as an antibodyhaving an affinity for a contaminant, unused reagent, reactionside-product or other compound unwanted in a final product. In the useof disks comprising such a filter-limited chamber, a fluid containing amixture of wanted and unwanted compounds is moved through the filterchamber by centripetal force of the rotating disk. The unwantedcompounds are thus bound by the affinity material, and the desiredcompounds flushed free of the chamber by fluid flow motivated bycentripetal force. Alternatively, the desired compound may be retainedin such a filter-limited chamber, and the unwanted compounds flushedaway. In these embodiments, egress from the chamber, for example by theopening of a valve, is provided.

3. Mixers

A variety of mixing elements are advantageously included in embodimentsof the Microsystems disks of the invention that require mixing ofcomponents in a reaction chamber upon addition from a reagent reservoir.Static mixers can be incorporated into fluid handling structures of thedisk by applying a textured surface to the microchannels or chamberscomposing the mixer. Two or more channels can be joined at a position onthe disk and their components mixed together by hydrodynamic activityimparted upon them by the textured surface of the mixing channel orchamber and the action of centripetal force imparted by the rotatingdisk. Mixing can also be accomplished by rapidly changing the directionof rotation and by physically agitating the disk by systems external tothe disk.

In other embodiments, flex plate-wave (FPW) devices (see White, 1991,U.S. Pat. No. 5,006,749, ibid.) can be used to effect mixing of fluidson a disk of the invention. FPW devices utilize aluminum andpiezoelectric zinc oxide transducers placed at either end of a very thinmembrane. The transducers launch and detect acoustic plate waves thatare propagated along the membrane. The stiffness and mass per unit areaof the membrane determine the velocity of plate wave. When connectedwith an amplifier, the waves form a delay-line oscillation that isproportional to the acoustic wave velocity. Structures based on the FPWphenomena have been used to sense pressure, acceleration, organicchemical vapors, the adsorption of proteins, the density and viscosityof liquids as well as to mix liquids together. FPW devices can beintegrated onto the disk or can be positioned in proximity to the diskto effect mixing of fluid components in particular reaction chambers onthe disk.

4. Valving Mechanisms

Control of fluid movement and transfer on the disk typically includesthe use of valving mechanisms (microvalves) to permit or prevent fluidmovement between components. Examples of such microvalves include apiezo activator comprising a glass plate sandwiched between two siliconwafers, as described by Nakagawa et al. (1990, Proc. IEEE Workshop ofMicro Electro Mechanical Systems, Napa Valley, Calif. pp. 89); aschematic diagram of such a valve is shown in FIG. 6. In thisembodiment, a lower wafer and glass plate can have one or two inlets andone outlet channel etched in them. An upper wafer can have a circularcenter platform and a concentric platform surrounding it. The base ofpiezoelectric stack can be placed onto the center platform and its topconnected to the concentric platform by means of circular bridge. Thecenter of a SiO₂/SiN₄ arch-like structure is connected to the piezoelement. Valve seats are made of nickel or other sealing substance. In athree-way embodiment, fluid moves from the center inlet port to theoutlet with no applied voltage. With a voltage applied the piezo elementpresses down on the arch center causing the ends to lift, blocking thecenter inlet and allowing fluid to flow from the peripheral inlet. Inother, two-way embodiments, fluid flows with no applied voltage and isrestrained upon the application of voltage. In another embodiment of atwo-way valve, fluid is restrained in the absence of an applied voltageand is allowed to flow upon application of a voltage. In any of theseembodiments the piezo stack can be perpendicular to the plane ofrotation, oblique to the plane of rotation, or held within the plane ofrotation.

In another embodiment, fluid control is effected using apneumatically-actuated microvalve wherein a fluid channel is etched inone layer of material that has a raised valve seat at the point ofcontrol (a schematic diagram of this type of valve is shown in FIG. 7).Into another layer, a corresponding hole is drilled, preferably by alaser to achieve a hole with a sufficiently small diameter, therebyproviding pneumatic access. Onto that second structure a layer ofsilicone rubber or other flexible material is spun-deposited. Thesestructures are then bonded together. Fluid movement is interrupted bythe application of air pressure which presses the flexible membrane downonto the raised valve seat. This type of valve has been described byVeider et al. (1995, Eurosensors IX, pp. 284-286, Stockholm, Sweden,June 25-29). Measurements made by Veider et al. have shown that asimilar valve closes completely with the application of 30 KPa ofpressure over the fluid inlet pressure. This value corresponds to 207psig, and can be adjusted by changing the diameter of the pneumaticorifice and the thickness of the membrane layer. Pneumatic pressure isapplied to the disk to activate such valves as shown schematically inFIG. 8.

Pneumatic actuation can also be embodied by a micromachined gas valvethat utilizes a bimetallic actuator mechanism, as shown in FIG. 9. Thevalve consists of a diaphragm actuator that mates to the valve body. Theactuator can contain integral resistive elements that heat uponapplication of a voltage, causing a deflection in the diaphragm. Thisdeflection causes a central structure in the actuator to impinge uponthe valve opening, thus regulating the flow of fluid through theopening. These valves allow proportional control based on voltage input,typically 0-15 V DC. These types of valve are commercially available(Redwood Microsystems, Menlo Park, Calif.; ICSensors, Milpitas, Calif.).

Embodiments of pneumatically actuated membrane valves can includeintegration of both components on a single disk or can comprise twodisks aligned so that the pneumatic outlets of one disk align with thesecond disk to impinge upon the pneumatic actuation orifice of the otherdisk. In either embodiment a source of pneumatic pressure can bedelivered to the disk via concentric rings of material such a Teflon®.In this embodiment, a standing core and a revolving element arecontiguous to the disk. Pneumatic pressure is delivered through theinterior of the standing core and directed by channels to the outer edgeof the standing core. Suitably placed channels are machined into therevolving element and impinge upon the channels in the standing core anddirect the pneumatic pressure to the gas valves.

Another valve embodiment is a pressure-balanced microvalve, shown inFIG. 10. This type of valve is constructed of three layers of material,comprising two layers of silicon separated by a thin layer ofelectrically-insulating oxide (i.e., silicon dioxide). A glass layer isbonded onto the top of the valve and advantageously contains inlet andoutlet ports. A center plunger-fashioned in the middle silicon layer isdeflected into a gap contained on the lower silicon layer by applicationof a voltage between the silicon layers. Alternatively, the plunger isdeflected by providing a pneumatic pressure drop into a gap in the lowerlayer. Irreversible jamming of micromachined parts may be prevented bythe application of a thin layer of Cr/Pt to the glass structure. As anelectrostatically driven device, this type of valve has many advantages,including that it may be wired directly in the fabrication of the disk.In this embodiment the actuator is a finely tuned device that requiresminimal input energy in order to open the valve even at relatively highpressures. These types of valves have been disclosed by Huff et al.(1994, 7′ International Conference on Solid-State Sensors and Actuators,pp. 98-101).

Another type of single-use valve, termed a polymeric relaxation valve,compatible with the disk and fluidic devices in general, is disclosedherein and shown in FIG. 11. This valve is based on the relaxation ofnon-equilibrium polymeric structures. This phenomenon is observed whenpolymers are stretched at temperatures below their glass transitiontemperature (T_(g)), resulting in a non-equilibrium structure. Uponheating above the T_(g), the polymer chains relax and contraction isobserved as the structure equilibrates. A common example of thisphenomenon is contraction of polyolefin (used in heat shrink tubing orwrap), the polyolefin structure of which is stable at room temperature.Upon heating to 135° C., however, the structure contracts. Examples ofPR valve polymers include but are not limited to polyolefins,polystyrenes, polyurethanes, poly(vinyl chloride) and certainfluoropolymers.

One way to manufacture a PR valve is to place a polymer sheet orlaminate over a channel requiring the valve (as shown in FIG. 11). Acylindrical valve is then cold-stamped in such a way as to block themicrochannel. The valve is actuated by the application of localizedheat, for example, by a laser or by contact with a resistive heatingelement. The valve then contracts and fluid flow is enabled.

A further type of microvalve useful in the disks of the invention is asingle use valve, illustrated herein by a capillary microvalve(disclosed in U.S. Provisional Application Ser. No. 60/023,756, filedAug. 12, 1996 and incorporated by reference herein). This type ofmicrovalve is based on the use of rotationally-induced fluid pressure toovercome capillary forces. Fluids which completely or partially wet thematerial of the microchannels (or reservoirs, reaction chambers,detection chambers, etc.) which contain them experience a resistance toflow when moving from a microchannel of narrow cross-section to one oflarger cross-section, while those fluids which do not wet thesematerials resist flowing from microchannels (or reservoirs, reactionchambers, detection chambers, etc.) of large cross-section to those withsmaller cross-section. This capillary pressure varies inversely with thesizes of the two microchannels (or reservoirs, reaction chambers,detection chambers, etc., or combinations thereof), the surface tensionof the fluid, and the contact angle of the fluid on the material of themicrochannels (or reservoirs, reaction chambers, detection chambers,etc.). Generally, the details of the cross-sectional shape are notimportant, but the dependence on cross-sectional dimension results inmicrochannels of dimension less than 500 μm exhibit significantcapillary pressure. By varying the intersection shapes, materials andcross-sectional areas of the components of the microsystems platform ofthe invention, “valve” are fashioned that require the application of aparticular pressure on the fluid to induce fluid flow. This pressure isapplied in the disks of the invention by rotation of the disk (which hasbeen shown above to vary with the square of the rotational frequency,with the radial position and with the extent of the fluid in the radialdirection). By varying capillary valve cross-sectional dimensions aswell as the position and extent along the radial direction of the fluidhandling components of the microsystem platforms of the invention,capillary valves are formed to release fluid flow in arotation-dependent manner, using rotation rates of from 100 rpm toseveral thousand rpm. This arrangement allows complex, multistep fluidprocesses to be carried out using a pre-determined, monotonic increasein rotational rate.

Control of the microvalves of the disks provided by the invention isachieved either using on-disk controller elements, device-specificcontrollers, or a combination thereof.

6. Control Systems

Integrated electronic processing systems (generally termed “controllers”herein) that include microprocessors and I/O devices can be fabricateddirectly onto the disk, can be fabricated separately and assembled intoor onto the disk, or can be placed completely off the disk, mostadvantageously as a component of the micromanipulation device. Thecontrollers can be used to control the rotation drive motor (both speed,duration and direction), system temperature, optics, data acquisition,analysis and storage, and to monitor the state of systems integral tothe disk. Examples of rotational controllers are those using rotationsensors adjacent to the motor itself for determining rotation rate, andmotor controller chips (e.g., Motorola MC33035) for driving directionand speed of such motors. Such sensors and chips are generally used in aclosed-loop configuration, using the sensor data to control rotation ofthe disk to a rotational set-point. Similarly, the rotational data fromthese sensors can be converted from a digital train of pulses into ananalog voltage using frequency-to-voltage conversion chips (e.g., TexanInstruments Model LM2917). In this case, the analog signal then providesfeedback to control an analog voltage set-point corresponding to thedesired rotation rate. Controllers may also use the data encoded in thedisk's data-carrying surface in a manner similar to that used incommercially-available compact disk (CD) players. In these embodiments,the digital data read by the laser is used to control rotation ratethrough a phase-locked loop. The rotation rate information inherent inthe frequency of data bits read may be converted to an analog voltage,as described above.

The controllers can also include communication components that allowaccess to external databases and modems for remote data transfer.Specifically, controllers can be integrated into optical read systems inorder to retrieve information contained on the disk, and to writeinformation generated by the analytic systems on the disk to opticaldata storage sections integral to the disk. In these embodiments it willbe understood that both read and write functions are performed on thesurface of the disk opposite to the surface comprising the Microsystemscomponents disclosed herein.

Information (i.e., both instructions and data, collectively termed“informatics”) pertaining to the control of any particular microanalyticsystem on the disk can be stored on the disk itself or externally, mostadvantageously by the microprocessor and/or memory of the disk device ofthe invention, or in a computer connected to the device. The informationis used by the controller to control the timing and open/closed state ofmicrovalves on the disk, to determine optimal disk rotational velocity,to control heating and cooling elements on the disk, to monitordetection systems, to integrate data generated by the disk and toimplement logic structures based on the data collected.

7. Power Supply

The electrical requirements of systems contained on a disk can bedelivered to the disk through brushes that impinge upon connectionsintegral to the disk. Alternatively, an electrical connection can bemade through the contact point between the microplatform and therotational spindle or hub connecting the disk to the rotationalmotivating means. A battery can be integrated into the disk to providean on-board electrical supply. Batteries can also be used to power thedevice used to manipulate the disk. Batteries used with the inventioncan be rechargeable such as a cadmium or lithium ion cell, orconventional lead-acid or alkaline cell.

Power delivered to the disk can be AC or DC. While electricalrequirements are determined by the particular assay system embodied onthe disk, voltage can range from microvolts through megavolts, morepreferably millivolts through kilovolts. Current can range frommicroamps to amperes. Electrical supply can be for component operationor can be used to control and direct on-disk electronics.

Alternatively, inductive current can be generated on the disk by virtueof its rotation, wherein current is provided by an induction loop or byelectrical brushes. Such current can be used to power devices on thedisk.

8. Detectors and Sensors

Detection systems for use on the microsystem platforms of the inventioninclude spectroscopic, electrochemical, physical, light scattering,radioactive, and mass spectroscopic detectors. Spectroscopic methodsusing these detectors encompass electronic spectroscopy (ultraviolet andvisible light absorbance, luminescence, and refractive index),vibrational spectroscopy (IR and Raman), and x-ray spectroscopies (x-rayfluorescence and conventional x-ray analysis using micromachined fieldemitters, such as those developed by the NASA Jet Propulsion Lab,Pasadena, Calif.).

General classes of detection and representative examples of each for usewith the microsystem platforms of the invention are described below. Theclasses are based on sensor type (light-based and electrochemical). Inaddition, the detection implementation systems utilizing the detectorsof the invention can be external to the platform, adjacent to it orintegral to the disk platform.

a. Spectroscopic Methods:

1. Fluorescence

Fluorescence detector systems developed for macroscopic uses are knownin the prior art and are adapted for use with the microsystem platformsof this invention. FIGS. 12A and 12B illustrate two representativefluorescence configurations. In FIG. 12A, an excitation source such as alaser is focused on an optically-transparent section of the disk. Lightfrom any analytically-useful portion of the electromagnetic spectrum canbe coupled with a disk material that is specifically transparent tolight of a particular wavelength, permitting spectral properties of thelight to be determined by the product or reagent occupying the reservoirinterrogated by illumination with light. Alternatively, the selection oflight at a particular wavelength can be paired with a material havinggeometries and refractive index properties resulting in total internalreflection of the illuminating light. This enables either detection ofmaterial on the surface of the disk through evanescent lightpropagation, or multiple reflections through the sample itself, whichincreases the path length considerably.

Configurations appropriate for evanescent wave systems are shown in FIG.12A (see Glass et al., 1987, Appl. Optics 26: 2181-2187). Fluorescenceis coupled back into a waveguide on the disk, thereby increasing theefficiency of detection. In these embodiments, the optical componentpreceding the detector can include a dispersive element t6 permitspectral resolution. Fluorescence excitation can also be increasedthrough multiple reflections from surfaces in the device whenever noisedoes not scale with path length in the same way as with signal.

Another type of fluorescence detection configuration is shown in FIG.12B. Light of both the fluorescence excitation wavelength and theemitted light wavelength are guided through one face of the device. Anangle of 90 degrees is used to separate the excitation and collectionoptical trains. It is also possible to use other angles, including 0degrees, whereby the excitation and emitted light travels colinearly. Aslong as the source light can be distinguished from the fluorescencesignal, any optical geometry can be used. Optical windows suitable forspectroscopic measurement and transparent to the wavelengths used areincluded at appropriate positions (i.e., in “read” reservoir embodimentsof detecting chambers) on the disk. The use of this type of fluorescencein macroscopic systems has been disclosed by Haab et al. (1995, Anal.Chem. 67: 3253-3260).

2. Absorbance Detection

Absorbance measurements can be used to detect any analyte that changesthe intensity of transmitted light by specifically absorbing energy(direct absorbance) or by changing the absorbance of another componentin the system (indirect absorbance). Optical path geometry is designedto ensure that the absorbance detector is focused on a light pathreceiving the maximum amount of transmitted light from the illuminatedsample. Both the light source and the detector can be positionedexternal to the disk, adjacent to the disk and moved in synchrony withit, or integral to the disk itself. The sample chamber on the disk canconstitute a cuvette that is illuminated and transmitted light detectedin a single pass or in multiple passes, particularly when used with astroboscopic light signal that illuminates the detection chamber to afrequency equal to the frequency of rotation or multiples thereof.Alternatively, the sample chamber can be a planar waveguide, wherein theanalyte interacts on the face of the waveguide and light absorbance isthe result of attenuated total internal reflection (i.e., the analytereduces the intensity source light if the analyte is sequestered at thesurface of the sample chamber, using, for example, specific binding to acompound embedded or attached to the chamber surface; see Dessy, 1989,Anal. Chem. 61: 2191).

Indirect absorbance can be used with the same optical design. Forindirect absorbance measurements, the analyte does not absorb the sourcelight; instead, a drop in absorbance of a secondary material is measuredas the analyte displaces it in the sample chamber. Increasedtransmittance therefore corresponds to analyte concentration.

3. Vibrational Spectroscopy

Vibration spectroscopic detection means are also provided to generatedata from a detecting chamber or “read” section of a microplatform ofthe invention. Infrared (IF) optical design is analogous to the designparameters disclosed above with regard to absorbance spectroscopy in theUV and visible range of the electromagnetic spectrum, with thecomponents optimized instead for infrared frequencies. For suchoptimization, all materials in the optical path must transmit IR light.Configuration of the optical components to provide Raman lightscattering information are similar to those disclosed in FIGS. 12A and12B above for fluorescent measurements. However, due to the illuminationtime needed to generate sufficient signal, the rotation rate of the diskmust be slowed, or in some instances, stopped. Depending on the use,static IR or Raman scattering analysis is most advantageously performedoff-line in a separate IR or Raman instrument adapted for analysis ofthe disks of the invention.

4. Light Scattering

Turbidity can also be measured on the disk. Optics are configured aswith absorbance measurements. In this analysis, the intensity of thetransmitted light is related to the concentration of the light-scatteredparticles in a sample. An example of an application of this type ofdetection method is a particle agglutination assay. Larger particlessediment in a rotating disk more rapidly than smaller particles, and theturbidity of a solution in the sample chamber before and after spinningthe disk can be related to the size of the particles in the chamber. Ifsmall particles are induced to aggregate only in the presence of ananalyte, then turbidity measurements can be used to specifically detectthe presence of an analyte in the sample chamber. For example, smallparticles can be coated with an antibody to an analyte, resulting inaggregation of the particles in the presence of the analyte as antibodyfrom more than one particle bind to the analyte. When the disk is spunafter this interaction occurs, sample chambers containing analyte willbe less turbid that sample chambers not containing analyte. This systemcan be calibrated with standard amounts of analyte to provide a gauge ofanalyte concentration related to the turbidity of the sample under a setof standardized conditions.

Other types of light scattering detection methods are provided for usewith the microsystems platforms and devices of the invention.Monochromatic light from a light source, advantageously a laser lightsource, is directed across the cross-sectional area of a flow channel onthe disk. Light scattered by particles in a sample, such as cells, iscollected at several angles over the illuminated portion of the channel(see Rosenzweig et al., 1994, Anal. Chem. 66: 1771-1776). Data reductionis optimally programmed directly into the device based on standards suchas appropriately-sized beads to relate the signal into interpretableresults. Using a calibrated set of such beads, fine discriminationbetween particles of different sizes can be obtained. Anotherapplication for this system is flow cytometry, cell counting, cellsorting and cellular biological analysis and testing, includingchemotherapeutic sensitivity and toxicology.

b. Electrochemical Detection Methods

Electrochemical detection requires contact between the sensor elementand the sample, or between sensor elements and a material such as a gasin equilibrium with the sample. In the case of direct contact betweensample and detector, the electrode system is built directly onto thedisk, attached to the disk before rotation or moved into contact withthe disk after it has stopped rotating. Detectors constructed using agas vapor to encode information about the sample can be made with thedetector external to the disk provided the gas vapor is configured tocontact both the sample chamber and the detector. Electrochemicaldetectors interfaced with the disk include potentiometric, voltammetricand amperimetric devices, and can include any electrochemical transducercompatible with the materials used to construct the microsystem disk.

1. Electric Potential Measurement

One type of electrochemical detection means useful with the Microsystemsplatforms of the invention is an electrical potential measurementsystem. Such a system provides a means for characterizing interfacialproperties of solutions passed over differently activated flow channelsin the instrument. In view of the temperature-controlled nature of themicroplatforms of the invention, streaming potentials can also bemeasured on this device (see Reijenga et al., 1983, J Chromatogr. 260:241). To produce streaming potentials, the voltage potential differencebetween two platinum leads in contact with a solution at the inner andouter portions of the disk is measured in comparison with a referenceelectrode. As fluid flows under controlled centripetal motion throughthe channel, a streaming potential develops in response to fluidinteractions with the disk surfaces in a moving field.

Alternatively, a platinum electrode is used to generateelectroluminescent ions (see Blackburn et al., 37: 1534-9).Chemiluminescence is then detected using one of the optical detectorsdescribed above, depending on the wavelength of the chemiluminescentsignal. Voltametric components are also useful in microsyntheticplatforms of the invention to produce reactive intermediates orproducts.

2. Electrochemical Sensors

Electrochemical sensors are also advantageously incorporated into thedisk. In one embodiment, an electrochemical detector is provided thatuses a redox cycling reaction (see Aoki et al., Rev. Polarogr. 36: 67).This embodiment utilizes an interdigitated microarray electrode within amicromachined chamber containing a species of interest. The potential ofone electrode is set at the oxidized potential of the species ofinterest and the potential of the other electrode is set at thereduction potential of the species of interest. This is accomplishedusing a dual channel potentiostat, allowing the oxidized and reduced(i.e., redox) chemical state of the sample to be determined, or thechamber may be preset for a particular species. A volume of fluidcontaining a substance of interest is directed to the chamber. Theelectrochemically reversible species is then oxidized and reduced bycyclically energizing the electrodes. In this embodiment a molecule isdetected by an apparent increase in the redox current. Sincenon-reversible species do not contribute signal after the first cycle,their overall contribution to the final signal is suppressed. Dataanalysis software is used to suppress signal due to non-reversiblespecies.

In another embodiment, a multichannel electrochemical detector isprovided comprising up to 16 lines of an electrode fabricated in achamber by photolithography with dimensions resulting in each line being100 μm wide with 50 μm between lines. (see Aoki et al., 1992, Anal.Chem. 62: 2206). In this embodiment, a volume of fluid containing asubstance of interest is directed to the chamber. Within the chambereach electrode is set a different potential so that 16 separate channelsof electrochemical measurement may be made. Additionally, each electrodepotential can be swept stepwise by a function generator. This protocolyields information pertaining to redox potential as well as redoxcurrent of the substances. This type of analysis also allowsidentification of molecules via voltammogram.

c. Physical Methods

Physical detection methods are also provided for use with the disks ofthe invention. For example, the disk can be used as a viscometer.Microchannels containing fluid to be tested advantageously contain abead inserted on the disk. The motion of the bead through the fluid isanalyzed and converted into viscosity data based on standards developedand stored in microprocessor memory. (see Linliu et al., 1994, Rev. SciInstrum. 65: 3824-28).

Another embodiment is a capacitive pressure sensor (see Esashi et al.,1992, Proc. Micro Electro Mechanical Systems 11: 43). In thisembodiment, silicon and glass substrates are anodically bonded withhermetically sealed reference cavities. Pressure may be detected by thecapacitance change between the silicon diaphragm and an aluminumelectrode formed on the glass. A capacitance-to-frequency converteroutput of a CMOS circuit can be integrated on the silicon substrate orcontained in controlling electronics off the disk.

By judicious placement of pressure sensors, the pressure due tocentrifugation can be determined at any position on the disk. Inconjunction with the microchannel diameter information and the patternof orientation of the channels on the disk, pressure data can be used todetermine flow rates at a particular rotational speed. This informationcan then be used by the microprocessor to adjust disk rotational speedto control fluid movement on the disk.

Surface acoustic wave (SAW) devices are also provided as components ofthe Microsystems platforms of the invention. These devices can be placedabove the disk to detect head-space gases, or incorporated in the fluidchannel on the instrument. When placed in the fluid system, the SAW isused to detect density changes in the solution, indicative of changingbuffer, reagent or reactant composition (see Ballantine et al., 1989,Anal. Chem. 61: 1989).

Volatile gases on the disk or trapped in the head-space surrounding thedisk can be monitored in several ways. For example, a Clark electrodepositioned in contact with either the solution of the gases above thedisk may be used to detect oxygen content. (Collison et al., 1990, Anal.Chem. 62: 1990).

d. Radioactive Detection Components

Microsystems platforms of the invention also can incorporateradioactivity detectors. Radioactive decay of an analyte or syntheticproduct on a disk of the invention can be detected using a CCD chip orsimilar single channel photodiode detector capable of integrating signalover time. Alternatively, radioactivity can be determined directly byplacing a solid state detector in contact with a radioactive analyte.(see Lamture et al., 1994, Nucleic Acids Res. 22: 2121-2125).

Modular Structures

Analytic systems provided as components of the platforms of theinvention typically consist of combinations of controllers, detectors,buffer and reagent reservoirs, chambers, microchannels, microvalves,heaters, filters, mixers, sensors, and other components. Components thatconstitute an analytic system on the disk can be composed of one or moreof the following: complete integral systems fabricated entirely on thedisk; complete integral systems fabricated as a component and assembledinto or onto the disk; a subset of components fabricated directly ontothe disk and interfaced with a subset of components that are fabricatedas a component and assembled into or onto the disk; components thatinterface with the disk externally through a synchronously spinningdisk; and components that interface with the spinning disk from aposition that remains stationary in relation to the disk (e.g., therotational spindle).

Methods and Uses

Because of its flexibility, the invention offers a myriad of possibleapplications and embodiments. Certain features will be common to mostembodiments, however. These features include sample collection; sampleapplication to disk, incorporating tests of adequacy at the time ofsample application; a variety of specific assays performed on the disk;data collection, processing and analysis; data transmission and storage,either to memory, to a section of the disk, or to a remote station usingcommunications software; data output to the user (including printing andscreen display); and sample disk disposal (including, if necessary, disksterilization).

Sample or analyte is collected using means appropriate for theparticular sample. Blood, for example, is collected in vacuum tubes in ahospital or laboratory setting, and using a lancet for home or consumeruse. Urine can be collected into a sterile container and applied to thedisk using conventional liquid-transfer technology. Saliva is preferablyapplied to the disk diluted with a small volume of a solution ofdistilled water, mild detergent and sugar flavoring. This solution canbe provided as a mouthwash/gargle for detecting antigens, biologicalsecretions and microorganisms. Alternatively, a small sack made of afishnet polymer material containing the detergent formulation and achewable resin can be chewed by a user to promote salivation, and thenremoved from the mouth and saliva recovered and applied conventionally.Amniotic fluid and cerebrospinal fluid are, of necessity, collectedusing accepted medical techniques by qualified personnel.

Environmental and industrial samples are collected from ground water orfactory effluent into containers produced to avoid leaching contaminantsin the sample. Soil samples are collected and mixed with a solventdesigned to dissolve the analyte of interest. Industrial applications,such as pyrogen screening, are accomplished using specially-designedsample ports.

Sample or analyte is loaded onto the disk by the user. Sample isoptimally loaded onto the disk at a position proximal to the center ofrotation, thereby permitting the greatest amount of centripetal force tobe applied to the sample, and providing the most extensive path acrossthe surface of the disk, to maximize the number, length or arrangementof fluid-handling components available to interact with the sample.Multiple samples can be applied to the disk using a multiple loadingdevice as shown in FIGS. 13A through 13C. In this embodiment of amultiple loading device, a multiplicity of pipette barrels are equallyspaced and arranged radially. The pipettes are spaced to provide thatthe tips of the pipettes fit into access ports on the surface of thedisk. The tips can be simple pins that hold a characteristic volume ofsample by virtue of a combination of surface properties and fluidcharacteristics. Alternatively, the tips can be conventional hollowtubes, such as capillary or plastic conical tips, and the fluidmanipulated manually in response to positive or negative pressure, aswith a manual or automatic pipetting device. The loader can be operatedmanually or by robotic systems. The barrels can also be arrayed in aflexible arrangement, permitting the tips to address a linear array inone configuration and a radial array in another. In each embodiment, theloader comprises an alignment device to ensure reproducible placement ofthe loading tips on the disks of the invention.

Loaders are designed specifically for the substances being investigated.Examples include medical uses (where the samples include blood, bodyfluids including amniotic fluid, cerebrospinal, pleural, pericardial,peritoneal, seminal and synovial fluid, in addition to blood, sweat,saliva, urine and tears, and tissue samples, and excreta), andenvironmental and industrial substances (including atmospheric gases,water and aqueous solutions, industrial chemicals, and soils). Loadingdevices are also advantageously compatible with standard blood-handlingequipment, such a vacuum tubes fitted with septa, and access sampletherein by piercing the septa. Loading devices are also compatible withseat collection devices and means, such as lancets, for obtaining asmall blood sample. A disk may also have integral lancets and rubberseals in order to sample blood directly.

Dynamic as well as static loading of the disk is envisioned as beingwithin the scope of the invention (see Burtis et al., 1974, Clin. Chem.20: 932-941).

As the invention comprises the combination of a Microsystems platform asdescribed above and a micromanipulation device for manipulating thisplatform to impart centripetal force on fluids on the platform to effectmovement, arrangement of components can be chosen to be positioned onthe disk, on the device, or both. Mechanical, electronic,optico-electronic, magnetic, magneto-optic, and other devices may becontained within the disk or on disk surface. Some on-disk devices havebeen described above in detail; additionally, the disk may containelectronic circuitry, including microprocessors for coordination of diskfunctions, and devices for communication with the disk manipulationdevice or other devices. The disk optimally comprises detectors andsensors, or components of these devices and energy sources for variousdetection schemes (such as electric power supplies for electrochemicalsystems, electromagnetic radiation sources for spectroscopic systems),or materials, such as optically-transparent materials, that facilitateoperation of and data generation using such detectors and sensors;actuators, including mechanical, electrical, and electromagnetic devicesfor controlling fluid movement on the disk, including valves, channels,and other fluid compartments; communications and data handling devices,mediating communications between the disk and the player/reader device,using electromagnetic (laser, infra-red, radiofrequency, microwave),electrical, or other means; circuitry designed for controllingprocedures and processes on the disk, including systems diagnostics,assays protocols and analysis of assay data, These are provided in theform of ASICs or ROM which are programmed only at thepoint-of-manufacture; FPGA's EPROM, flash memory (UV-erasable EPROM), orprogrammable IC arrays, or similar arrays programmable by the userthrough the platform manipulation device or other device. Also includedin the components of the invention are CPU and microprocessor units andassociated RAM operating with an assembler language or high-levellanguage programmable through disk communications, and components formediating communication with other devices, including facsimile/modemcommunications with remote display or data analysis systems.

Off-disk devices comprise the microplatform micromanipulating deviceitself and other devices which can access information, writeinformation, or initiate processes on the disk. FIG. 15 illustrates thecategories of devices and sub-devices which are part of themicromanipulation device, and indicates how there components interact.“Interaction” is used herein to mean the exchange of “data” between thedisk and device, or among the components of the device itself. Therelationship between these components is here described, followed bydetailed examples of the components.

These include the mechanical drive and circuitry for rotation monitoringand control, overall system control, data read/write devices, externaldetectors and actuators for use with the disk, dedicated data and assayprocessors for processing encoded data and assay data, a centralprocessor unit, a user interface, and means for communicating to thedisk, the user, and other devices. Mechanical drive and associatedcircuits include devices to control and monitor precisely the rotationrate and angular position of the disk, and devices to select and mountmultiple-disks from a cassette, turntable, or other multiple-diskstorage unit. System control units provide overall device control,either pre-programmed or accessible to the user-interface. Disk dataread/write devices are provided for reading encoded information from adisk or other medium. Optimally, write-to-disk capabilities ateincluded, permitting a section of the disk to contain analytical datagenerated from assays performed on the disk. This option is notadvantageous in uses of the disk where the disks are contaminated withbiological or other hazards, absent means (such as sterilization) forneutralizing the hazard. The device can also include external actuatorscomprising optical magneto-optic, magnetic and electrical components toactuate microvalves and initiate processes on the disk, as well asexternal detectors and sensors or components of detectors and sensorsthat operate in concert with other components on the disk, includinganalytic and diagnostic devices. Certain of these aspects of the diskmicromanipulating device are illustrated in FIGS. 14A through 14F.

Disk data processors are also advantageously incorporated into thedevices of the invention which enable processing and manipulation ofencoded disk data. These components include software used by themicromanipulator CPU, programmable circuits (such as FPGAs, PLAs) anddedicated chipsets (such as ASICs). Also provided are assay processorsfor processing data arising from events and assays performed on the diskand detected by external detectors or communicated from on-diskcomponents. The device also advantageously comprises a centralprocessing unit or computer which will allow processing of disk data andassay results data-analysis (through pre-programming); additionally,conventional computer capabilities (word-processing, graphicsproduction, etc.) can be provided.

A user interface, including keypads, light-pens, monitors, indicators,flat-panel displays, interface through communications options tohost-devices or peripheral devices, and printers, plotters, and graphicsdevices are provided as components of the microplatformmicromanipulating devices of the invention. Communication andtelecommunications are provided through standard hard-wired interfaces(such as RS-232, IEE-488M SCSI bus), infra-red and opticalcommunications, short-or long-range telecommunications (“cellular”telecommunications radio-frequency), and internal or external modem formanual or automated telephone communications.

Disk information comprises both software written to the disk tofacilitate operation of the microsystem assays constructed thereupon,and assay data generated during use of the microsystem by the user. Diskinformation includes material written to the disk (as optically encodeddata) and information inherent to the disk. (e.g., the current status ofa valve, which can be accessed through magnetic pickup or through thereflective properties of the coating material at the valve-position)Data written to the disk may include but is not limited to theaudio/video/test and machine format information (e.g., binary, binhex,assembler language). This data includes system control data used forinitiation of control programs to spin the disk, or perform assays,information on disk configuration, disk identity, uses, analysisprotocols and programming, protocols descriptions, diagnostic programsand test results, point-of-use information, analysis results data, andbackground information. Acquired data information can be stored asanalog or digital and can be raw data, processed data or a combinationof both.

System control data include synchronization data to enable themicromanipulation device to function at the correct angularvelocity/velocities and accelerations and data relating to physicalparameters of disk. Disk configuration and compatibility data includedata regarding the type of disk (configuration of on-disk devices,valves, and reagent, reaction and detection chambers) used to determinethe applicability of desired testing protocols; this data provides afunctional identity of the type of disk and capabilities of the disk. Itcan also form part of an interactive feedback system for checkingmicrosystem platform components prior to initiation of an assay on thedisk. Disk identify and serial numbers are provided encoded on each diskto enable exact identification of a disk by fabrication date, disk typeand uses, which data are encoded by the manufacturer, and userinformation, which is written to the disk by the user. Also included indisk data is a history of procedures performed with the disk by theuser. Also included in the disk data is a history of proceduresperformed with the disk, typically written for both machine recognition(i.e., how many and which assays remain unused or ready for use), aswell as information written by the user.

FIGS. 30-32 display the action of software encoded on the disk used forcontrolling the device driving the disk. Figure 0.30 displays theprocess flow. The control program, encoded as data on the disk, is readthrough conventional means, for example, by the laser of an opticalstorage medium (such as a compact disc or “Laservision” disc) anddecoded in the conventional way for loading into the random accessmemory (RAM) of the micromanipulation device. This program is thenexecuted. In some applications, execution of the program to completionwill be automatic and without active interaction with the user. In otherapplications the user will be presented with a variety of options(typically, as a menu) for running the program. As an example, userchoices, such as whether to run an exhaustive or limited set ofdiagnostics, test procedures, analyses, or other disk functions, or todetermine the extent of detail and the method of reporting test resultsare provided through the user interfaces.

FIGS. 31 and 32 show one specific set of programmed steps for performingassays using the capillary microvalves disclosed above; otherarrangements of steps within the program will be apparent to one ofordinary skill and readily integrated, for example, for sending signalsto activate microvalves and other actuators. The program disclosed hereconsists of blocks in which different rotation rates are set for varyingamounts of time, allowing for capillary valving, mixing, and incubation;mixing program blocks, which (for example) put the spindle motor throughan oscillatory acceleration and deceleration, are possible but notshown. These program blocks consist of outputting commands to variouselectronic devices (motor, detectors, etc.) and reading data fromdevices, yielding a measure of device and process status. Provisions areshown in the program for halting the program if the status is “bad”(such as motor cannot reach appropriate speed, door to device cannotclose, no power detected in light source for spectroscopicmeasurements). This condition can lead to a program halt (as shown) orsend the program back to the user for further instructions via theinterface.

The program shown here additionally incorporates data acquisition, dataanalysis, and data output blocks. The particular acquisition processhere involves using an encoded signal on the disk—for example, anoptical signal associated with a detection chamber passing thedetector—to gate acquisition of data. In this way, data is acquired fora specific time when detection chambers are in proximity to thedetector. It is also possible to continuously take data and use featuresin that data—for example, the shape of the signal as a function of time,which might look like a square wave for an array of windows on anotherwise opaque disk—to determine what parts of the data are useful foranalysis. Data analysis could include non-linear least-squares fitting,linear regression of data as function of time, or end-point analysis(data at an en d-point time for a reaction), as well as other methods.Data output may be in the form of “yes/no” answers to the userinterface, numeric data, and storage to internal or external storagemedia

All component parts of this program need not be contained on the disk.For example, the program can be resident in the computer and designed toread the disk itself to obtain the rotation velocity profiles necessaryfor using the disk. All other aspects of the program—such as when andhow to read and analyze data—an be part of a dedicated program or readfrom other media.

Analysis/test protocol data are descriptions of tests and analyses whichcan be performed with a disk. These data can be a simple as a titlegiven the disk, or can contain a detailed description of disk use, dataanalysis and handling, including test protocols and data analysisprotocols. Analysis/test protocol programming is provided that can beused as systems-specified subroutines in more general software schemes,or can be fed into programmable logic so that the device can perform thedesired analyses. Analysis/protocol descriptions are provided, as audio,video, text or other descriptions of analytic processes performed ondisk, including background information, conditions for valid use,precautions, and other aspects.

Encryption and verification data/programming is provided to ensure thesecurity of the programming and data generated in the analyses performedby the disk. Encryption/de-encryption routines are used to restrictedaccess to data contained on the disk. Such-routines also used in medicaldiagnostic applications.,

System self-diagnostics are also provided. System diagnostics includediagnostic test results on detector function, status of reagentchambers, valves, heating elements, and other components, stored indisk-memory or written to the disk by a separate device used at the timeof diagnostics.

Point-of-use information is encoded on the disk at its point-of-use(sample loading, e.g.) in the form of video, audio, or text images,including, for example location, time and personnel. Also included inpoint of use information is test result data, recorded by the diskitself or by a disk player/reader at the time these procedures wereperformed.

Certain data are inherent to the disk and are accessible through themicromanipulation device. These include sample adequacy test data, whichrecords the presence or absence of samples or reagents at appropriatereservoirs and other fluid handling regions of the disk, and can beaccessed through external detectors and sensors. Valve status is alsorecorded, including the record of the change in valve status during aprocedure performed in the disk. Valve status is determined, forexample, by using magnetic pickups in the device applied to magneticvalve mechanisms; status can also be visible through optical windows onthe disk. The presence of radioactive, chemical or biologicalcontaminants on the external surface of the disk can be recorded upondetection by sensors comprising the device, optimally resulting in awarning message delivered to a user interface such as a display orprint-out.

Disk data and information are stored using a variety of media, includingboth the recording medium of the disk material (i.e., reflectiveproperties of an optically-read disk, most preferably a read/writeCD-ROM) and by the device itself using electronic components.Information is encoded using conventional or modified technologies usedfor computer information storage. Video, audio, and text information isdigitized using methods developed by the digital video, audio, andcomputer industries. Analog signals arising from test procedures, suchas a signal observed in a photodiode detector or photomultiplier tube,are converted through analog-to-digital conversion regimes or may besupplied in raw or amplified form through external jacks for processingoff-disk or off-device. Various embodiments of the disk manipulationdevice of the invention include the capacity to both read and write datato the disk or to use read-only data from any of these media types.Encryption and authentication codes can be used for security purposes.Disk data storage media include optical media, utilizingreflecting/non-reflecting flats and pits on a surface, using technologyadapted from audio CD, CD-ROM, and “Laserdisc” technology, and barcodes.Magnetooptic and magnetic media are also within the scope of this aspectof the invention, using conventional computer magnetic storage media.Electronic data storage means are also provided, using the status ofinternal arrays of electronic components (FPGAs, PLAs, EPROM, ROM,ASICs, IC networks) for information handling. Chemical recording means,including simple chromatographic staining of a detector section orchamber of the device, is also disclosed to provide a simple visualrecord of a test result. This simple chemical recording means providesan avenue to at-home diagnostic without the need for an expensive devicemore sophisticated in capabilities than required to determine an assayamenable to simply the presence or absence of chemical markers.

Software and Communications

Software providing the information and instruction set for microsystemperformance, quality control, data acquisition, handling and processing,and communications is included within the scope of this invention. Forthe purposes of this invention, such software is referred to as “machinelanguage instructions.” Control and analysis software is advantageouslyprovided in high-level languages such as C/C++, Visual Basic, FORTRAN orPascal. Drivers are provided for interface boards (either internal tothe device or to a host computer interfaced with the device) whichtranslates instructions on the host computer's bus into micromanipulatorcommands. Additionally, drivers for experiment-control software such asLabView may be created, again using conventional, industry-standardinterface protocols. These applications are most preferably capable ofbeing run on a number of popular computer platforms, includingUNIX/Linux, X-windows, Macintosh, SGI, etc.

Control and analysis can also be performed using dedicated chipsets andcircuitry, ROM, and EPROM. For example, test validity can be insured (atleast in part) through the use of ROM-based test procedures, in whichall programming is performed at the point-of-manufacture withoutpossibility of end-user corruption. Separate application software canalso be developed so that data from a disk-player can be analyzed onnon-controller platforms, using available applications (such as Excel,Clarisworks, SigmaPlot, Oracle, Sybase, etc.).

Because some applications of the disk technology disclosed hereininvolve important questions related to human health, disk diagnosticsoftware must be able to analyze diagnostics of the disk, its contents(samples, reagents, devices), the player, and analysis software toensure result validity. Types of information used by this diagnosticsoftware include sample adequacy and flow, verification of disk formatand software/test procedure compatibility, on-and off-disk softwaretests, quality control monitoring of disk manufacture (for example,channel placement and alignment), viability, positioning andfunctionality of on-disk and off-disk sensors and detectors, diagnosticsof player communications and microprocessor, microprocessor/CPU, powerstability, etc.

Diagnostics of mechanical and electronic components are performed inways familiar to those proficient in the art. Software self-diagnosticsare achieved using checklist/verification of software routines andsubroutines to detect incompatibility with system hardware (from eitherthe micromanipulation device or the disk) or with other components ofsystem software.

Sample-related disk diagnostics include assays of flow, sample adequacy,and reagent adequacy, type and quality for the assay to be performed.Device-related disk diagnostics include checks of detector/sensorfunction, electronic components self-test, valve control, and thermalcontrol tests. Software diagnostics provide self-testing of softwarecomponents encoded in the disk or in the device, corruption safeguards,read-only and read-write tests. Disk format is also checked using diskdiagnostics, ensuring that the disk format and assay type are properlyread and are in agreement with the protocol held in the device memory.

On-disk software includes read-only software, available as ROM,specifically CD-ROM, for diagnostics, assay control and data analysis.Read-only software is designed for specific procedures and processeswhich cannot be altered and insure proper usage of the disk andfail-safe against corruption by the user. Software may also be embodiedwithin the encoding medium (optical, magnetic, etc.) or an alternatemedium (such as barcodes). Re-programmable software (such as FPGAs,PLAs, EPROMs, or IC arrays) can be re-programmned by the diskmicromanipulation device or devices designed for this purpose. Similartypes of software are alternatively provided on-device. In either case,a user-interface through keyboard, touchpad and/or display components ofthe device is provided.

Applications software is provided in read-only or re-programmablesoftware formats. Included in this component of the fluidicsmicromanipulation apparatus of the invention is software that can beread from standard computer data storage medium Examples include medicalor analytic diagnostic programs reliant on integrated data-bases whichare contained within disk or device memory, or that can be accessed fromnetworked workstations, or access on-line services, such as a newsletterand news services, and software for the production and analysis ofimages, including pattern recognition, statistical analysis software,etc.

Integration of control and applications software can be made through theuse of either a unique operating system developed for the disk andmicromanipulator of the invention, or by adaptation of existing OS.Optimally, the OS uses authoring software to combine text, graphics,video and audio into an easy to use, “point and click” system. Such asOS could also provide an object-oriented environment or facsimilethereof (e.g., LabView-based systems) for customizing programming bysophisticated users, as well as providing for the development ofadditional software by the disk reader/player manufacturer orindependent software developers.

The OS can also be chosen to allow design of disks and disk-basedassays. Mechanical design, including simulation of rotational dynamicsand stability and fluid flow simulation are advantageously encompassedin a disk design software package.

Communications aspects of the invention include hardware and softwareembodiments relating to data input and output from a user or to remotecontrol and analysis sites. Hard-wired communications features includehigh-speed data-, video- or image-transmission and communication throughlocal busses (e.g., a VGA bus for video signals) and conventionalhard-wired interfaces (e.g., RS-232, IEEE488, SCSI bus), Ethernetconnections, Appletalk, and various local area networks (LANs).Telecommunications devices include cellular transceivers for short-rangecommunications, radio-frequency and micro-wave transceivers forlong-range communications, and internal or external modem for manual orautomated telephone communications. Video in/out ports, analog out-linesfor data transmission, input jacks for input of analog signals fromother instruments, and optical and infra-red communications ports arealso provided for communications with peripheral instruments.

Configurations of the Fluidics Micromanipulation Apparatus for CertainApplications

The micromanipulation device includes various combinations of hardwareand software as described above. FIG. 15 is an illustration of thegeneral combination of communication, device, detection, and controlinstrumentation in a device. Certain applications may not have certainfeatures, for example, portable units may not have graphical userinterfaces. The micromanipulation device can be a “stand-alone” device,or a peripheral instrument to a larger assemblage of devices including,for example, computers, printers, and image-processing equipment, or ahost for peripheral elements such as control pads, data entry/read-outunits (such as Newton-type devices or equivalent), or an integratedsystem. The device in all embodiments comprises hardware to rotate thedisk at both steady and variable rates and systems for monitoringrotation rate. The device can also include devices to initiate sampleand disk diagnostics, perform “external”tests and detection as describedherein, initiate sample and disk diagnostics, perform “external” testsand detection as described herein, initiate analyses on-disk throughspecific actuators such as valves, read disk-inherent information andinformation encoded in the disk or other data/information storage mediainformation, and in some applications write information to the disk.

Additional elements in the device, including system control, dataprocessors, array of assay processors, external detectors, externalactuators, assay out and data out lines, communications, and software,are device-and/or application-specific.

For example, in a “point-of-use” portable or home-use application,sample loading is followed by initiation of the player's program. Systemcontrol can be provided by front-panel controls and indicators which canaccess a variety of programs stored in the disk or the device. These“hard-wired” programs utilize controller circuitry to read or read/writeoperations from or to disk or memory, and/or perform tests usingexternal devices. The device can be designed for performance of a singleprocedure, or can be pre-programmed to perform a set of procedures ormultiple embodiments of the same procedure using a single disk. Deviceactuation is optimally obtained with the pressing of a single button.These processor(s) and data processors(s) of this type of devicecomprise circuitry and chipware designed to process analysis data (assayprocessor) and encoded data (data processor). Information from theseprocessors can be available for output to the user on a front-panel orvideo display and can also be used internally to ensure correctoperating conditions for the assay. This internal information processingcan include the results of systems diagnostic tests to insure diskidentity and test type compatibility; the presence of reagent and sampleas determined through light absorption through a detector port scanningreagent and sample reservoirs; the presence of contamination detectedbefore testing begins, and the results of self-diagnostics on externaldetectors and actuators. These results are used by the system controllerto determine whether the requested test can be performed.

After loading and activation, analysis results can be stored internallyin electronic memory or encoded upon the disk. The results of theseanalyses and procedures are then routed to the front-panel display(flat-panel LCD, etc.) using appropriate video drivers. Processed assaydata can also be routed to one of many standard digital I/O systemsincluding RS-232, RS-232C, IEEE-488, and other systems familiar fromdigital I/O and interface. Similarly, encoded disk data can be routed tothe audio/visual display. Raw analog signals can also be switched to oneor more external jacks for off-device storage or processing.

An embodiment of the least technically sophisticated device is aportable unit no larger than a portable audio CD player consisting ofdisk-drive, controllers and selectors for programmable or pre-programmedangular acceleration/decelerationprofiles for a limited number ofprocedures. Such a device is advantageous for on-sitetoxic-chemical/contamination testing. Analyte to be tested is introducedto the disk, which is inserted into the player and the appropriateprogram chosen. Analysis results are stored on the disk, to be laterread-out by a larger player/reader unit, and/or displayed immediately tothe user. Results can also be stored as the inherent state of anindicator (positive/negative status of litmus paper in differentcuvettes, for example), with no other data collection or analysisperformed by the device. This data would be accessed by a largerplayer/reader or by other means outside the field-work environment.Information about the location, time, and other conditions of samplecollection are entered through the user interface.

Another embodiment is a stand-alone device with active communicationscapabilities and greater functionality. An exemplary application forsuch a device is as a home blood-assay unit. This device is used by anindividual placing a drop of blood on the disk; inserting the disk, andinitiating the assay, preferably simply by pressing a single button. Oneor more analytical procedures are then performed. Assay data istransferred to software which performs the requisite analysis, eitheron-disk or within the device. The device can also be permanently ortemporarily attached to the home-telephone line and automaticallytransmit either raw or reduced data to a computer at the centrallocation is used to analyze the data transmitted, compare the data withaccepted standards and/or previous data from the same patient, make apermanent record as part of a patient's device a confirmation of receiptof the data, perhaps the data analysis, and advice orsuggested/recommended course of action (such as contacting thephysician).

A desk-top peripheral/host application station constitutes a device asdescribed above with the ability to accept instructions from and respondto a host computer over one of many possible data-protocols. The systemis capable of acting as host or can transmit data to peripherals orother networked devices and workstations. Remote accessing ofpre-programmed functions, function re-programming, and real-time controlcapabilities are also provided.

Yet another embodiment of this application is a centralized or bedsideplayer/reader device with associated software located as a nurses'station in a hospital. As tests are performed on disks, the informationis relayed to a physician by telephone, facsimile or pager viashort-range transceiver. Patient identity can be entered at the time ofsample collection by the use of bar codes and light pens attached to thedevice, providing the advantage of positive patient/sampleidentification.

The device can also be provided having the above-capabilities andfunctionality's and in addition having an interface with an integratedcomputer having high-resolution graphics, image-processing and otherfeatures. The computer provides control of the device for performing thefunctions described above for the peripheral system, while physicalintegration greatly increases data-transmission rates. Additionally, theintegrated system is provided with extensive analysis software andbackground data-bases and information. Disk-storage cassettes ofcarousals are also an advantageous feature of such system. An integratedsystem of this type is useful in a large, analytical laboratory setting.

A self-contained system is useful for applications in isolatedenvironments. Examples include devices used in remote or hostilesetting, such as air, water and soil testing devices used in the Arcticfor environmental purposes, or for use on the battlefield for toxicchemical detection.

The microsystem platforms provided by the invention are also useful forpreparing samples for other analytical instruments, such asmass-spectrometers, gas chromatographs, high pressure liquidchromatographs, liquid chromatographs, capillary electrophoresis,inductively-coupled plasma spectroscopy, and X-ray absorptionfine-structure. In some application, the final product is removed fromthe disk to be analyzed.

Samples can be pre-concentrated and purified on the device byincorporating aqueous two-phase separation systems. This can be done,for example, by mixing two phases which separate from each other basedon thermodynamic differences like polyethylene glycol (PEG) anddextrans; biopolymers are usefully separated using this method.Alternatively, environmental tests such as calorimetric analysis can beenhanced by incorporating cloud-point separations to concentrate andenhance optical signals. In addition, small scale counter-currentchromatography can be performed on the device (see, Foucault, 1991,Anal. Chem. 63: PAGE). Centripetal force on the disk can be used toforce different density fluids to flow against each other, resulting inseparation of components along a density gradient to develop thechromatogram.

Applications and Uses

The microsystem platforms and micromanipulating devices that make up thefluidics micromanipulation apparatus of the invention have a widevariety of microsynthetic and microanalytic applications, due to theflexibility of the design, wherein fluids are motivated on the platformby centripetal force that arises when the platform is rotated. Whatfollows is a short, representative sample of the types of applicationsencompasses within the scope of the instant invention that is neitherexhaustive or intended to be limiting of all of the embodiments of thisinvention.

The invention is advantageously used for microanalysis in research,especially biological research applications. Such microanalyses includeimmunoassay, in vitro amplification routines, including polymerase chainreaction, ligase chain reaction and magnetic chain reaction. Molecularand microbiological assays, including restriction enzyme digestion ofDNA and DNA fragment size separation/fractionation can also beaccomplished using the microsystem disks of the invention.Microsynthetic manipulations, such as DNA fragment ligation, replacementsynthesis, radiolabeling and fluorescent or antigenic labeling can alsobe performed using the disks of the invention. Nucleic acid sequencing,using a variety of synthetic protocols using enzymatic replacementsynthesis of DNA, can be performed, and resolution and analysis of theresulting nested set of single-stranded DNA fragments can be separatedon the disk, identified and arranged into a sequence using residentsoftware modified from such software currently available formacroscopic, automated DNA sequencing machines. Other applicationsinclude pH measurement, filtration and ultralfiltration, chromatography,including affinity chromatography and reverse-phase chromatography,electrophoresis, microbiological applications including microculture andidentification ofpathogens, flow cytometry, immunoassay and otherheretofore conventional laboratory procedures performed at a macroscopicscale.

An illustrative example is immunoassay. While there exist a multiplicityof experimental methodologies for detecting antigen/antibodyinteractions that are in research and clinical use at the present time,the most robust immunoassay protocols involve “sandwich”-type assays. Insuch assays, an immobilized antibody is presented to a sample to betested for the antigenic analyte specific for the immobilized antibody.A second antibody, specific for a different epitope of the same antigenis subsequently bound, making a “sandwich” of the antigen between thetwo bound antibodies. In such assays, the second antibody is linked to adetectable moiety, such as a radiolabel or fluorescent label, or aenzymatic or catalytic functionality. For example, horseradishperoxidase or alkaline phosphatase are used to produce a color change ina substrate, the intensity of which is related to the amount of thesecond antibody bound in the sandwich.

An example of a disk adapted for performing such an immunoassay is shownin FIG. 17Q. In this embodiment, the secondary antibody is linked toalkaline phosphate (AP). The presence and amount of AP activity isdetermined by monitoring the conversion of one of the followingexemplary substrates by the enzyme calorimetrically: B-naphthylphosphate converts to an insoluble azo dye in the presence of adiazonium salt; 5-bromo-4-chloro-3-indolyl phosphate is converted to5,5′-dibromo-4-,4′-dichloro indigo in the presence of cupric sulfate; or4-methylumbelliferyl phosphate is converted to 4-methylumbelliferone,which emits light at 450 nm.

In one exemplary embodiment, the reaction chamber comprises an antibodyspecific for an antigen, where the antibody is immobilized by adsorptionof the antibody to the reaction chamber. Contiguous with the reactionchamber is advantageously placed a reagent reservoir containing a secondantibody, this antibody being liked to an enzyme such as alkalinephosphate. Sample, which may contain an antigen of interest that isspecifically recognized by the above antibodies, is loaded at an inletport. The disk is spun to first introduce the sample into the reactionchamber containing immobilized antibody, followed by introduction of thesecond antibody into the reaction chamber after a time sufficient tosaturate the immobilized antibody with antigen to the extent the antigenis present in the sample, Alternatively, the sample may be contactedwith the second antibody, allowed to interact, then introduced into thereaction chamber. Incubation of the sample with antibody is performedwithout spinning for about 1 minute. After each incubation, washingbuffer from a buffer reservoir is spun into the reaction chamber inorder to remove unbound antibody. For alkaline phosphatase assays,solutions of 2 mg/mL o-dianisidine in water, 1 mg/mL B-naphthylphosphate in 50 mM boric acid/50 mM KCl (pH 9.2) buffer and 100 mMmagnesium chloride are delivered to the reaction chamber in theappropriate amounts. The extent of enzyme-linked, secondary antibodybinding is evaluated by detection of a purple precipitate using aphotodiode or CCD camera.

A disk configured for immunoassay applications is shown in FIG. 17R forillustration.

In an alternative embodiment of the immunological assays of theinvention, the invention provides a means for identifying andquantitating the presence and number of particular cells or cell typesin fluids, most preferably biological fluids such as blood, urine,amniotic fluid, semen and milk. In these embodiments of the invention,the Microsystems platform comprises a chamber or solid surface on thedisk that is prepared to selectively bind the particular cell or celltype. After attachment of the cells to the surface, non-specific bindingcells and other components are removed by fluid flow (washing) orcentrifugal force (comprising the inertial flow of fluid in response tothe centripetal acceleration of the disk). The cells of interest thatremain attached to the microplatform surface or chamber are the detectedand quantified using means including but not limited to microscopic,spectroscopic, fluorescent, chemiluminescent, or light-scattering means.The invention also provides such cells attached to a specific surfacefor toxicity monitoring, such as metabolic monitoring to determine theefficacy of bioactive drugs or other treatments. Ordered arrays of suchsurface are provided in certain embodiments to facilitate a completedetermination of the purity and sterility of certain biological samples,and for cell cytometric and cytometry applications.

The surface or chamber of the disk for specific binding of theparticular cells or cell types of interest is prepared to providespecific binding sites therefor. Typically, an antibody, preferably amonoclonal antibody, is attached to the surface or chamber, wherein theantibody is specific for a cell surface antigen expressed on the cell orcell type of interest. Alteratively, a ligand specific for a cellsurface receptor expressed on the particular cell or cell type ofinterest is used to provide a specific attachment site. Arrays ofspecifically prepared surfaces or chambers are provided on certainembodiments of the disk. Surfaces and chamber are provided, for example,by contacting the surface with a solution of an appropriate antibody. Inthe practice of these preparation methods, contact of the surface withthe antibody is followed by contacting the surface with a non-specificblocking protein, such as bovine serum albumin. Antibodies and blockingproteins can be contacted with the surface or chamber using apiezoelectrically driven point head (such as are used in ink-jetprinting applications) can be advantageously used for this purpose.Alternatively, screen printing, or spraying the antibody solution on thechamber or surface using an airbrush can be employed. These methods arepreferred in preparing surfaces and chambers in the 0.1-10 mm scale. Inadditional alternatives, microlithographic and microstamping techniquescan be used to prepare the surface or chamber.

In the practice of the invention, a biological or other fluid samplecontaining the particular cell or cell type of interest is applied tothe prepared surface or chamber and allowed in contact with the preparedsurface or chamber for a time sufficient to allow specific binding ofthe cells or cell types to the surface. As contact with the surface maybe inhibited by cell settling properties in the volume of the fluid,chambers and surfaces having minimized height transversely through themicrosystem platform are preferred.

Non-specific cell binding is minimized or eliminated from the chamber orsurface by washing the surface or chamber with a fluid amount sufficientto remove such non-specific binding. Washing is accomplished by simplebulk flow of fluid over the surface or chamber, or by centrifugation.

After washing, cells that remain attached to the surface or chamber aredetected and counted. In a preferred embodiment, detection and countingis achieved using fluorescence microscopy. In the practice of theinvention, specific dyes can be used to provide a fluorescence signalfor any live cells remaining of the disk. The dye can be added directlyto the surface or chamber, for example using a membrane-permeant dye,such as acetoxy-methyl ester dyes. Alternatively, specific antibodiescan be linked to such dyes. Dyes can be added to the biological fluidcomprising the cells prior to introduction onto the microsystemplatform, or such dyes can be contacted with the cells in situ on thedisk. The presence of the cells is detected using a fluorescencedetector comprising a light source, a source filter, a dichroic filteror, mirror, an emission filter, and a detector such as a photomultipliertube.

In another example, thin-layer chromatography is accomplished on amicroplatform disk comprising 100 pm square cross-section channelsradiating outward from the center of the disk. Each channel is filledwith separation substrate, which typically contains a binder material(0.1-10%) such as starch, gypsum, polyacrylic acid salts and the like,to provide mechanical strength and stability. (The use of such compoundsin conventional TLC applications is discussed in Poole et al., 1994Anal. Chem. 66: 27A). Sorbents are also included in the materialscomprising the separation channels, including for example cellulose,polyamide, polyethylene powders, aluminum oxide, diatomeceous earth,magnesium silicate, and silica gels. Such substrates can be modified forexample with silanizing molecules, such as dimethyl-, ethyl-octa- and3-aminoprophy-silanes. Preferentially the separation substrate containssorbent-impregnated fiber glass or PFTE matrices.

Sample is loaded via a port located proximal to the center of rotationof the disk. Upon spinning the disk, a mobile phase is allowed to flowoutward through the separation substrate, carrying sample components tothe periphery of the disk at characteristic rates. The mobile phase canbe chosen from a multiplicity of appropriate solvent systems includinghexane, methanol and dichloromethane. Choice of a particular solventdepends on the nature of the disk material, the separation substrate andthe components of the sample to be separated. Similarly, the choice ofvisualization reagents used to detect separated sample components arespecific for the substances separated. For example, ninhydrin is used todetect amino acids; alimony chloride is used plus potassium permanganatefor hydrocarbons; sulfuric acid plus anisaldehyde for carbohydrates; andbromine for olefins. Imagine of separation channels after separation isachieved using a CCD camera. A disk configured for him layerchromatography applications is shown in FIG. 17R for illustration.

Medical applications using the Microsystems of the invention areabundant and robust. Various embodiments of the invention provide forat-home, bedside, hospital and portable devices for rapid analysis ofblood components, blood gases, drug concentrations, metabolities andinfectious agents. In at-home monitoring embodiments, the inventionprovides a simple, easy-to-use consumer friendly device requiring apatient to add a blood droplet, urine sample or saliva sample to aspecific application region on the disk, insert the disk in the deviceand start the device by pushing a button. In a hospital setting, bothbedside and clinical laboratory embodiments are provided, wherein thebedside embodiment is advantageously linked electronically to a centralprocessing unit located, for example, at a nurses station, and theclinical laboratory embodiment comprises a medical reference library forrapid, automated diagnostics of patient sample. The medical applicationsof the instant invention include blood testing (such as monitoringplatelet counts in patients being treated with chemotherapeutic drugs);immunoassay for metabolites, drugs, and other biological and otherchemical species; vaccine efficacy monitoring; myeloma or lupuserythematosus monitoring; determination of blood glucose and/or ketonebody levels in patients with diabetes; automated cholesterol testing:automated blood drug concentration determination; toxicology; monitoringof electrolytes of other medically-relevant blood component at apatient's bedside; sepsis/endotoxin monitoring; allergy testing; andthrombus monitoring.

The invention also provides analytical instruments for environmentaltesting, industrial applications and regulation compliance. Portable,preferably hand-held embodiments, as well as more extensive embodiments,installed as part of an industrial quality control regime, are provided.Applications for these embodiments of the invention include analytetesting, particularly testing for industrial effluents and wastematerial, to be used for regulatory compliance; and quality control ofindustrial, most advantageously of human consumable items, particularlypharmaceuticals and specifically endotoxin determinations. Applicationfor testing, mixing and evaluating perfumes and other complex mixturesare also within the scope of the invention.

The invention also provides chemical reaction and synthesis modeling,wherein a reaction scheme or industrial production regime can be testedand evaluated in miniaturized simulations. The invention provides forcost-effective prototyping of potential research, medical and industrialchemical reaction schemes, which can be scaled to macroscopic levelsafter analysis and optimization using the microsystems platforms of thisinvention.

A variety of other applications are provided, including microsyntheticmethods and forensic applications.

The following Examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.

EXAMPLE 1 Fabrication of Microplatform Disks for Chemical Analysis,Synthesis, and Applications

Microplatform disks of the invention are fabricated from thermoplasticssuch as Teflon, polyethylene, polypropylene, methylmethacrylates andpolycarbonates, among others, due to their ease of molding, stamping andmilling. Alternatively, the disks can be made of silica, glass, quartzor inert metal. A fluid handling system is built by sequentialapplication of one or more of these materials laid down in stepwisefashion onto the thermoplastic substrate. FIGS. 17A through 17E are aschematic representation of a disk adapted for performing DNAsequencing. Disks of the invention are fabricated with an injectionmolded, optically-clear base layer having optical pits in the manner ofa conventional compact disk (CD). The disk is a round, polycarbonatedisk 120 nm in diameter and 100 pm thick. The optical pits provide meansfor encoding instrument control programming, user interface information,graphics and sound specific to the application and driver configuration.The driver configuration depends on whether the micromanipulation deviceis a hand-held, benchtop or floor model, and also on the details ofexternal communication and other specifics of the hardwareconfiguration. This layer is then overlaid with a reflective surface,with appropriate windows for external detectors, specifically opticaldetectors, being left clear on the disk. Other layers of polycarbonateof varying thickness are laid down on the disk in the form of channels,reservoirs, reaction chambers and other structures, including provisionson the disk for valves and other control elements. These layers can bepre-fabricated and cut with the appropriate geometries for a givenapplication and assembled on the disk. Layers comprising materials otherthan polycarbonate can also be incorporated into the disk. Thecomposition of the layers on the disk depend in large part on thespecific application and the requirements of chemical compatibility withthe reagents to be used with the disk. Electrical layers can beincorporated in disks requiring electric circuits, such aselectrophoresis applications and electrically-controlled valves. Controldevices, such as valves, integrated circuits, laser diodes, photodiodesand resistive networks that can form selective heating areas or flexiblelogic structures can be incorporated into appropriately wired recesses,either by direct fabrication of modular installation onto the disk.Reagents that can be stored dry can be introduced into appropriate openchambers by spraying into reservoirs using means similar to inkjetprinting heads, and then dried on the disk. A top layer comprisingaccess ports and air vents, ports or shafts is then applied. Liquidreagents are then injected into the appropriate reservoirs, followed byapplication of a protective cover layer comprising a thin plastic film.

A variety of other disk configurations are disclosed in FIGS. 17Fthrough 17P, adapted for particular applications as described in theFigure legends.

EXAMPLE2 Blood Composition Determination

Blood composition can be determined via hematocrit analysis using ananalytic microplatform disk prepared as described in Example 1 heldwithin a device comprising a microchannel layer with a number ofmicrochannels as shown in FIG. 18. The microchannel layer is 100 pmthick and treated with heparin to prevent coagulation during the assay.The blood sample to be analyzed is drawn by capillary action into achannel arranged perpendicular to the direction of rotation, as shown inFIG. 18; a number of such channels may be arranged radially on the disk.When all samples to be tested have been drawn into the channels, thedisk is spun at a speed of 8000 to 10,000 rpm to effect sedimentation oferythrocytes within the channel. Once centrifugation has been performedfor an appropriate time (3 to 5 minutes), the hematocrit of each sampleis determined simultaneously by stroboscopic interrogation of each ofthe channels using a conventional CD laser system in the devicedescribed above. When the laser passes the boundary of erythrocytes, thechange in light scattering pattern detected by the photodiode detectoris converted into a hematocrit value based on a standardized set oflight scatter/hematocrit information stored in the internal processorand memory of the device. Alternatively, the raw information is relayedvia a infrared port or hard-wired interface to a microprocessor foranalysis. Such a central microprocessor is on site or in the alternativeat a centralized location, such as a nursing station in a hospital or ina medical center connected to the hematocrit determining device bytelephone or other dedicated connection. Hematocrit can be determined byuntrained individuals (including patients) by the simple application ofa blood droplet produced by lancet onto the disk, followed by the simpleapplication of the device and automated hematocrit analysis and dataprocessing on site or transmission to a central location of trainedmedical personnel. This embodiment of the invention provides for chronicmonitoring of patients having hematopoietic proliferative disease (suchas leukemia, lymphoma, myeloma, and anemias).

In addition, blood gas can be determined using the above device incombination with a disk having integrated electrodes embedded within thehematocrit channel, or having a separate channel devoted to blood gasdetermination on the hematocrit disk. Blood oxygenation (P0₂) isdetermined by a Clark-type electrode consisting of a thin Cr—Au cathodeand an Ag—AgCl wire anode. The amount of carbon dioxide in the blood isdetermined by a Severing-type electrode using an ISFET (a type of fieldeffect transistor) as a pH monitor. Blood pH is determined with the useof a Sl₃N₄ gate ISFET with a reference electrode consisting of a liquidjunction and an Ag—AgCl wire electrode. Further examples of suchanalytical methods for determining blood gases, electrolyteconcentration and other information advantageously performed using thehematocrit disk or alternate variations of this disk are described asmodifications of the macroscopic-scale methods of Shoji & Esashi (1992,Sensors and Actuators B 8: 205).

Blood analysis are also performed using split-flow thin cell (SPLITT)fractionation as described by Bor Fuh et al. (1995, Biotechnol. Prog.11: 14-20). A schematic representation of a disk configured for SPLITTanalysis is shown in FIG. 19. This process can produce enrichedfractions of proteins and lipoproteins, platelets, erythrocytes,lymphocytes, monocytes, and neutrophils. A non-contiguous circularchannel is etched into the disk incorporating a thin wall at either end(FIG. 19), the inlet stream splitter. Sample and carrier streams areintroduced at opposite sides of one end, and the chamber is spun in thatdirection. Within the spinning chamber two distinct splitting planes areset up based on hydrodynamic forces, the inlet splitting stream (ISP)and the outlet splitting stream (OSP). The ISP is adjustable byregulating the ratio of the sample to the carrier streams. Depending onthe method of sample input two distinct separation modes are possible,the equilibrium and transport modes.

In the equilibrium mode separation is based on the equilibrium of thecomponents in relation to the applied centrifugal field. Separation isoptimized by adjusting the outlet flow ratio. The enriched fraction canthen be collected from either side of the outlet stream splitter. In thetransport mode the components are introduced as a thin lamina above theISP. Based on the difference in sedimentation coefficients componentswith a higher transport rate are selectively directed to the oppositesides of the outlet valves at the orifices. Variable flow valves aredescribed elsewhere in this document. In another embodiment each SPLITTchamber may be dedicated to the separation type required of it, ISP orOSP, and the flow regulated by fixed flow-restriction orifices.

In order to fully fractionate blood into the above-identified fractions,five separations, each yielding two fractions, are performed. Oneembodiment of the Microsystems disk of the invention used for this typeof fractionation is shown in FIG. 19. Five concentric SPLITT cells areillustrated in this Figure, labeled C1, (close to the center ofrotation) through C5 (toward the periphery). A blood sample isintroduced into C 1 and subjected to a transport mode separation byrotating the disk at the appropriate speed. Platelets and proteins(fraction 1) are fractionated toward the center of rotation and bloodcells (fraction 2) move toward the periphery. Fraction 1 is routed tothe inlet of C2 while fraction 2 is routed to C3 by the opening andclosing of appropriately-positioned valves on the disk. The fractionsare then subjected to transport and equilibrium mode separationsrespectively. Using these techniques, Fraction 1 results in plateletstoward the center of rotation and proteins toward the periphery.Fraction 1 results in platelets toward the center of rotation andproteins toward the periphery. Fraction 2 yields fractions 3 and 4,consisting of lymphocytes and monocytes toward the center of rotationand erythrocytes and neutrophils toward the center of rotation andmonocytes toward the periphery. Fraction 4 yields neutrophils toward thecenter of rotation and erythrocytes toward the periphery. Thus,fractionation of blood into five isolated components is achieved.

The activity of enzymes in the protein fraction can be determining usingimmobilized enzymes (Heineman, 1993, App. Biochem. Biotech. 41: 87-97).For example, blood-specific enzymes (such as glucose oxidase, alkalinephosphatase, and lactate oxidase) can be immobilized in poly (vinylalcohol (PVAL). Lactate oxidase is immobilized on platinized graphiteelectrodes by sandwiching a thin layer of enzyme between two layers ofPVAL. The sensor responds to lactate by the electrochemical oxidation ofhydrogen peroxide generated by the enzyme-catalyzed oxidation of lactatethat diffuses into the network. The current produced is proportional tothe concentration of peroxide, which in turn is proportional to theconcentration of lactate. This sensor has been shown to be sensitive tolactate concentrations ranging form 1.7-26 μM.

Upon separation, each fraction is interrogated by detection systems todetermine the relative components of the fractions. Alternatively, eachfraction can be removed from the disk through an outlet port for furtherstudy off-device. For example, each fraction can be subjected to simplecounting by passing the cells in a thin stream past two electrodescomprising a resistance monitor. As a cell passes through the electrodesa corresponding rise in resistance is monitored and counted. These dataare then integrated relative to a standard set of particles distributedaccording to size to determine the relative number of each cell type inthe original sample.

The fractions can be subjected to fluorescent antibody staining specificto each cell type. The cells are held in place by micromachined filtersintegral to the channels (U.S. Pat. No. 5,304,487), stained and washedon the disk. The resulting labeled cells can then be quantified as afunction of the degree of fluorescent staining associated with thecells.

EXAMPLE 3 DNA Sizing and Mutation Detection

DNA sizing and detection of specific mutations in DNA at a particularsite are carried out using double stranded melting analysis with a diskprepared according to Example 1 and illustrated in FIG. 20. A DNAmeltometer (as described in co-owned and co-pending U.S. Ser. No.08/218,030, filed Mar. 24, 1994 and incorporated herein by reference inits entirety) is advantageously incorporated into the structure of thedisk Example 1. The DNA meltometer technique takes advantage of the factthat the denaturing point of a DNA duplex is dependent upon the length,the base composition, and the degree of complimentarity of the twostrands in the duplex. A denaturing point may be determined in relationto some physical state of the molecule (such as temperature or theconcentration of a denaturing chemical such as urea or formamide, and aset of standard conditions employed, the information derived from whichcan be stored in the microprocessor and/or memory of the device. Inorder to size any particular DNA duplex, one strand is immobilized onthe disk by attaching it to a streptavidin coated bead. The bead isretained by a filter machined in to the channel (see U.S. Pat. No.5,304,487). Alternatively, the bead can be a paramagnetic bead retainedin the channel by application of a magnetic filed using a permanentmagnet incorporated into the disk of positioned in proximity to thechannel. An electromagnet can be used. The electromagnet can beincorporated directly into the disk and actuated by application of 0.8volt DC at 500 mA. The other strand is labeled, typically using afluorescent dye or a radioactive isotope. Alternatively, the distinctoptical properties of the DNA molecule itself (i.e., hyperchromicity)are detected using unlabeled DNA molecules by monitoring absorbance at260 nm. Although this aspect of the method requires a more sophisticateddevice to generate and detect ultraviolet light, user preparation of theDNA is minimized and the cost of DNA preparation per sample greatlyreduced. In the practice of the method of the invention, theimmobilized, labeled duplex is placed on the disk and subjected to aflow stream of a buffered solution contained on the disk. During thedevelopment of the flow stream, the DNA is further subjected to acontrolled denaturing gradient produced in the flow stream by thegradual addition of denaturant to the DNA. With an effective radius of3.5″ and a rotational speed of 600 rpm, a flow rate of 10 uL/min can begenerated in a channel 10 μm in diameter. Four buffer reservoirs eachcontaining 300 uL can be incorporated into each quadrant of the disk(800 um deep extending from a position at a radius of 25 mm to 50 mm).At 10 uL/min, this will allow a melting ramp of 30 min. Each duplexdissociates at a characteristic concentration of denaturant in thegradient, and can be identified in comparison with standards thedenaturant profile information of which is stored in the microprocessorand/or memory of the device. Denaturation is detected by interrogationdownstream of the melting chamber, using the appropriate detecting means(photooptical means for ultraviolet absorption or fluorescencedetection, or radioisotope detectors (Geiger-Mueller counters) for DNAstands labeled with radioisotopes).

Exemplary of the uses the disks and devices of this aspect of theinvention is the detection, identification and size determination of DNAfragments produced by polymerase chain reaction or magnetic chainreaction (the latter disclosed in U.S. Ser. No. 08/375,226, filed Jan.19, 1995, which is a file wrapper continuation of U.S. Ser. No.08/074,345, filed Jun. 9, 1993 and 08/353,573, filed Dec. 8, 1994, eachincorporated by reference in its entirety). Amplification is carried outusing one primer labeled with a detectable label such as a fluorescentdye or radioisotope, and the other primer is covalently attached to amolecule that permits immobilization of the primer (e.g., biotin). Afteramplification (either off-disk or on the disk as described in moredetail in Example 4 below), the labeled, biotinylated duplex DNA productfragment is attached to a solid support coated with streptavidin, forexample, by movement of the amplification reaction mixture into achannel or compartment on the disk wherein the walls are coated withstreptavidin, or by movement of the amplification mixture into acompartment on the disk containing a binding matrix such as Dynal -280Dynabeads (polystyrene coated paramagnetic particles of 2.8 um indiameter). Standardized size markers are included in thepost-amplification compartment in order to provide a reference set ofDNA fragments for comparison with the amplification product fragments.In this analysis, a number of different duplex DNA molecules from eithera multiplex amplification reaction or a number of separate amplificationreactions may be sized simultaneously, each fragment or set of fragmentsbeing distinguished from others by use of reaction- or fragment-specificdetectable labels, or differences in some other physical property of thefragments. For amplifications performed off-disk, beads attached to thefragment are loaded into a channel on the disk capable of retaining thebeads (such as size exclusion, “optical tweezers” or by magneticattraction). In the latter embodiment, the magnetic retention means(permanent magnets or electromagnets) are either integral to the disk,held on second disk spinning synchronously with the first, or placed onthe device so as to immobilize the DNA fragments in the appropriatecompartment.

DNA size analysis is also performed essentially as described above,whereby the retained particles are subjected to a thermal denaturinggradient. For a thermal gradient used to denature the bound DNAfragments, a Peltier heat pump, direct laser heating or a resistiveelement is used to increase the temperature of the binding compartmentthrough the denaturation range by the gradual addition of thermalenergy. As above, a flow rate of 10 μL/min can be generated in a channel100 μm in diameter, allowing a melting ramp of 30 min. The compartmentis also subjected to a flow stream as described above to elute thedenatured, labeled stands from the binding/melting chamber. Downstreamfrom the binding/melting chamber are appropriate means for detecting DNAfragment denaturation, such as laser excitation at the resonantfrequency of the dye label and photodiode detection. The strength andcorresponding temperature of the raw absorbance or other signal isintegrated by the microprocessor and the size of each DNA fragmentdetermined by comparison to internal DNA size marker controls and DNAmelting profiles and characteristics stored in the microprocessor and/ormemory of the device.

DNA mutations are also detected by meltometer analysis. DNA fragments tobe tested (including amplification-derived fragments and restrictionsenzyme digestion or cloned fragments) are prepared and hybridized with abound standard (typically wildtype) copy of the gene or gene fragment ofinterest. Hybridization is performed either on-device or usingconventional DNA hybridization methods (as described in Hames & Higgins,Nucleic Acid Hebridization: A Practical Approach. Rickwood & Hames,eds., IRL Press: Oxford, 1985). Elution of the hybridized fragments isdependent on the degree of complimentarity between the two species ofDNA strands (i.e., wildtype and mutant). Hybridization analysis isperformed using wildtype DNA that is prepared wherein one strand iscovalently attached to a molecule that permits its immobilization. Thenon-covalently attached strand is then eluted by washing at atemperature much greater than the T_(m) of the duplex (typically, theDNA is heated to >90° C., or to lower temperatures in the presence ofdenaturants such as formamide). Elution is monitored to determine theconcentration of bound single-stranded product available for furtherhybridization; typically, the amount of DNA eluted is monitored, forexample by ultraviolet light absorbance, and the bound DNA considered tobe completely single stranded when no more DNA can be eluted. Thewildtype DNA is prepared whereby only one of the strand making up theduplex is covalently attached to the immobilizing molecule, in order torequire detectable labeling of only one (the complementary one) strandof the mutant DNA to be tested. Alternatively, either strand may becovalently attached, requiring both mutant strands to be detectablylabeled. An advantage of double-labeling the mutant fragment even whenonly one wildtype strand is covalently attached to the immobilizingmolecule, is that denaturation and elution of the non-complementarystrand can be monitored during hybridization, and non-specificbinding/hybridization of the mutant to wildtype DNA strands can bedetected.

After hybridization is accomplished, the degree of complementarity ofthe strands is determined by a modification of the thermal or chemicaldenaturing protocols described above. Analysis of the resulting patternof duplex melting is performed by comparison to a pattern of mismatchedDNA duplex melting prepared either simultaneously or prior toexperimental analysis and stored in the device microprocessor and/ormemory using standard or expected single base or multiple mismatches.Such comparison form the basis for a determination of the rapidscreening of individuals for a variety of characterizeddisease-associated genetic polymorphisms.

DNA mutations are also detected by meltometer analysis. In thisembodiment, test DNA is immobilized on the disk and subjected tohybridization/denaturation analysis with a battery of precharacterizedtest probes. Using this method, DNA fragments are preferably preparedusing in vitro amplification techniques, so that one strand isimmobilizable due to covalent attachment of the binding molecule to oneof the primers. Using this method, the DNA fragment to be tested issequentially hybridized with and eluted by denaturation from a series ofwell-characterized DNA probes being detectably labeled. Alternatively(depending on the nature of the DNA mismatch expected for each probe),hybridization and denaturation are multiplexed, using probes detectablylabeled with different detectable labels so that each probe can beidentified. This method is useful for genetic screening as describedabove.

EXAMPLE 4 DNA Amplification and Analysis

Fragments of DNA are amplified in vitro by polymerase chain reaction(PCR) or magnetic chain reaction and analyzed by capillaryelectrophoresis. Reagent mixing, primer annealing, extension anddenaturation in an amplification cycle resulting amplification of a 500bp target fragment and its subsequent analysis are carried out using adevice and disk as described in Example 1 above. A schematic diagram ofthe structure of the disk is shown in FIG. 21.

The disk comprises at least three sample input ports A, B and C. Port Apermits injection of 30 attomoles (about 100 pg) linear bacteriophagelambda DNA. Port B and C allow input of 5 μL of a 20 μM solution ofprimer 1 and 2 respectively, having the sequence: (SEQ ID No.: 1) Primer1: 5′GATGAGTTCGTGTCCGTACAACTGG-3′ and (SEQ ID No.: 2) Primer 2:5′-GGTTATCGAAATCAGCCACAGCGCC-3′.

The disk also comprises three reagent reservoirs D, E and F in theFigure and containing 54 μL of distilled water; 10 μL of a solution of100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂, 0.1% gelatin and 1.25μM of each dNTP; and 1 μL of Taq DNA polymerase at a concentration of 5Units/μL, respectively.

In addition, the disk comprises a reaction chamber G that is configuredto facilitate mixing of these reagents using a flexural-plate-wavecomponent (as described in U.S. Pat. No. 5,006,749). Also included inthe configuration of reaction chamber G are cooling and heating meansvia a Peltier component. These components can be integral to the disk orcan be positioned in the device so as to provide heating and coolingspecific for the reaction chamber. Disks are also provided that comprisea multiplicity of sets of the reaction components A through G.

Amplification is initiated by introducing sample DNA and primer intoeach set of ports A, B and C. When all samples and primers have beenintroduced into the ports, the disk is spun at a speed of 1 to 30,000rpm to effect mixing of the reagents into reaction chambers G.Simultaneously, valves controlling reservoirs D, E and F are opened andthe contents of these reservoirs are also forced into reaction chamberG. Mixing of sample DNAs, primers and reagents is facilitated byactivation of the flexural-plate-wave component. DNA amplification takesplace in the reaction chamber using the following thermocycling program.The reaction mixture is initially heated to 95° C. for 3 minutes. Theamplification cycle thereafter comprises the steps of: step 1,incubation at 95° C. for 1 minute; step 2, cooling the chamber to 37° C.for 1 minute; and step 3, heating the chamber to 72° C. for 3 minutes.This amplification cycle is repeated for a total of 20 cycles, and thereaction completed by incubation at 72° C. for 5 minutes.

Amplified DNA fragments are analyzed by transfer to capillaryelectrophoresis unit H by spinning the disk at a speed of 1 to 30,000rpm and opening a valve on reaction chamber G leading to capillaryelectrophoresis unit H, thereby effecting transfer of an amount of thereaction mixture to the electrophoresis unit. The amount of the reactionmixture, typically 10 μL, is determined by a combination of the lengthof time the valve on reaction chamber G is open and the speed at whichthe disk is rotated. Capillary electrophoresis is accomplished asdescribed below in Example 11, and fractionated DNA species detectedusing optical or other means as described above in Example 2. Thismethod provides a unified amplification and analysis deviceadvantageously used for performing PCR and other amplification reactionsin a sample under conditions of limited sample.

EXAMPLE 5 DNA Restriction and Digestion and Analysis

Restriction enzyme digestion and restriction fragment analysis isperformed using a disk and device as described above in Example 1. Adouble-stranded DNA fragment is digested with a restriction endonucleaseand subsequently analyzed by capillary electrophoresis. Reagent mixing,DNA digestion and restriction fragment analysis are carried out on thedisk. A schematic diagram of the structure of the disk is shown in FIG.22.

The disk comprises a sample input port A; three reagent reservoirs B, Cand D; a reaction chamber E configured for mixing the reagents asdescribed above in Example 5, and a capillary electrophoresis unit F.The reagent reservoirs contain: 1-2 μL of a restriction enzyme, e.g.HindIII, at a concentration of 20 Units/μL in reservoir B; 4 μL of asolution of 10 mM Tris-HCl (pH 7.9), 100 MM MgCl₂ and 10 mMdithiothreitol in reservoir C; and 30 μL of distilled water in reservoirD. Disks are also provided that comprise a multiplicity of sets of thereaction components A through E.

Restriction enzyme digestion of the DNA is initiated by placing 4-5 μLof a solution (typically, 10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 4μg bacteriophage lambda DNA in sample input port A. The DNA sample andthe reagents in reservoirs B, C and D are transferred to reactionchamber E by spinning the disk at a rotational speed of 1 to 30,000 rpmand opening valves controlling reservoirs B, C and D. The reaction isincubated at 37° C. for 1 h in reaction chamber E after mixing, thereaction chamber being heated by provision of a Peltier heating elementeither on the disk or positioned in the device so at to specificallyheat the reaction chamber. After digestion, an amount of the digestedDNA is transferred to electrophoresis unit F by spinning the disk at aspeed of 1 to 30,000 rpm and opening a valve on reaction chamber Eleading to capillary electrophoresis unit F, thereby effecting transferof an amount of the reaction mixture to the electrophoresis unit. Theamount of the reaction mixture, typically 10 μL, is determined by acombination of the length of time the valve on reaction chamber E isopen and the speed at which the disk is rotated. Capillaryelectrophoresis is accomplished as described below in Example 11, andfractionated DNA species detected using optical or other means asdescribed above in Example 2.

EXAMPLE6 DNA Synthesis

Oligonucleotide DNA synthesis is performed using a disk and device asdescribed above in Example 1. Synthesis is achieved by the stepwisetransport of controlled pore glass (CPG) through a series of reactionchambers containing reagents necessary for phosphoramidite DNAsynthesis. Reagents and CPG are delivered sequentially to reactionchambers by single-use valves connecting the reaction chambers to eachother and to reagent reservoirs. Each disk has a number of synthesisreaction chambers to produce oligonucleotides having a length similar tothe length of oligonucleotides produced by commercially-available DNAsynthesis instruments (i.e., 100-150 bases). A schematic diagram of thestructure of the disk is shown in FIG. 23A.

A CPG bearing a first base of a sequence (thereby defining the 3′ extentof the oligonucleotide) is loaded either by the user or by automatedmeans into a sample input port A. The CPG is then transferred into areaction chamber containing trichloroacetic acid (TCA) in acetonitrile(CH₃CN) by spinning the disk at a rotational speed of 1 to 30,000 rpm.Detritylation of the nucleotide is performed at room temperature for adefined time interval, typically 1 minute. The reagent is then decantedfrom the first reaction chamber by opening a valve with a bore too smallto allow passage of the CPG but sufficient to drain the TCA-containingmixture into a decantation chamber. As the deprotection of the base bydetritylation is known to produce a colored product (orange), theintensity of which is a measure of the extent of the reaction, opticalmeans for determining the absorbance of this effluent are advantageouslyprovided to be recorded on the device microprocessor/memory. Afterdecanting the reaction mixture, the CPG are spun into a rinse chambercontaining CH₃CN, the chamber optionally comprising a mixing means asdescribed above. After rinsing, the CH₃CN is decanted into a effluentreservoir controlled by a size-selective valve as above, and the CPGspun into a second reaction chamber. Mixed with the CPG in the secondreaction chamber is a solution containing one of four phosphoramiditebases (G, A, T, or C) corresponding to the next position in theoligonucleotide chain. The reaction mixture in the second reactionchamber is mixed and allowed to react for a defined time interval,typically three minutes. The reaction mixture is then decanted as aboveand the CPG spun into a rinse chamber containing CH₃CN and a mixingmeans. After rinsing, the CH₃CN is decanted to an effluent reservoir andthe CPG is spun into a third reaction chamber containing an oxidizingmixture of iodine, water, pyridine and tetrahydrofuran, where thereaction mixture is incubated for a defined time interval, typically 1minute. The reaction mixture is decanted to an effluent reservoir andthe CPG spun into a rinse chamber containing CH₃CN. After rinsing, theCH₃CN is decanted to an effluent reservoir and the CPG spun into afourth reaction chamber along with a two-component “capping” reagent.The capping reaction is performed for a defined time interval, typically1 minute. After the reaction is complete, the reaction mixture isdecanted to an effluent reservoir as above and the CPG spun into a rinsechamber containing CH₃CN. The CH₃CN is then decanted to an effluentreservoir and the CPG is spun into a fifth chamber containing TCA,comprising the beginning of another cycle. The cycle is repeated bytransit of the CPG through interconnected series of the four reactionchamber until the preprogrammed sequence is completely synthesized. TheCPG is then spun into a reaction chamber containing concentratedammonium hydroxide and heated at 60° C. for a defined time interval,typically 6 hours, during which time the DNA molecule is deprotected andcleaved from the CPG support. The finished oligonucleotide is removed bythe user or by automated means.

The disk provides a series of reaction chambers linked to each other andcomprising four reaction and rinsing chambers per nucleotide to be addedto the oligonucleotide chain. The disks can be loaded to produce aparticular oligonucleotide, or each reaction chamber 2 can be in contactwith reagent reservoirs containing each of the four nucleotide bases andlinked to the reaction chamber by an individually-controllable valve. Inthis embodiment, activation of the appropriate valve at each step in thecycle is controlled by a signal from the device. Disks comprising amultiplicity of these synthetic arrays. Permitting simultaneoussynthesis of a plurality of oligonucleotides, are also provided. Aschematic diagram of a disk configured for multiple oligonucleotidesynthesis is shown in FIG. 23B.

DNA synthesis can also be performed upon preloaded CPG contained inreaction chambers toward the periphery of the disk and reagentsdelivered by the use of multiuse two-way valves, as schematicallydiagramed in FIG. 23A. In these disks, reaction chambers capable ofcontaining. 100 nL, spaced 150 μm on-center (measured from the center ofone sphere to the center of the next sphere) in a disk of a 120 mmdiameter, as many as 1250 reaction chambers can be manufactured. Reagentreservoirs containing sufficient volumes to supply the reagent chamberson the disk are prefilled with the four phophoramidites, CH₃CN, TCA,oxidizer and capping reagents. Trityl-bearing CPG or linkers bounddirectly to the reaction chambers are similarly preloaded onto the disk.Microliter volumes of reagents are sufficient for each reaction. TCA isspun into each first reaction chamber and allowed to react for a definedlength of time, typically one minute, then spun to a effluent (waste)chamber on the periphery of the disk. The CH₃CN rinse is spun into eachreaction chamber and then to waste. By selective valve actuation, the A,C, G, or T phosphoramidite is spun to the reaction chambers requiringthat base and reacted for a defined time interval, typically threeminutes, and the spun to waste. A CH₃CN rinse is spun to each reactionchamber and after, to the waste chamber. The oxidizer mixture is spuninto each reaction chamber, reacted for a defined time interval,typically one minute, then to waste. Another CH₃CN rinse is spun to eachreaction chamber and then to waste. The two-component capping reagent isspun to each reaction chamber and reacted for a defined time interval,typically one minute, then to waste. For each cycle, the final CH₃CNrinse is then spun to each reaction chamber and then to the wastechamber. The cycle is repeated for a preprogrammed number of cyclesuntil each oligonucleotide is completely synthesized. Concentratedammonium hydroxide is then spun to each of the reaction chambers andreacted for a defined length of time, typically 6 hours, and reacted at60° C. to deprotect and cleave the completed DNA from its support. TheDNA can then be removed by manual or automated means. Conversely, thelinkage of the oligonucleotide to the CPG support is chosen to beresistant to the action of ammonium hydroxide, so that the deprotectedoligonucleotide remains in the reaction chambers bound to CPG.

Peptide synthesis disks are also provided, whereby the arrangement ofreagent reservoirs and reaction chambers as described above is adaptedfor the synthetic reactions comprising a peptide synthesis regime.

EXAMPLE 7 Enzymatic DNA Sequencing

The nucleotide sequence of a DNA fragment is determined by the Sangerenzymatic sequencing method using a disk prepared as described inExample 1 above (see FIG. 24). Template DNA (200 pg in 250 mL) and 100femtomoles of an appropriate primer are pipetted manually or by anautomated process into a sample input port. The DNA is then transferredinto a mixing chamber containing terminator solution (i.e., a solutioncomprising a dideoxy form of nucleotides G, A, T or C) by spinning thedisk at a rotational speed of 1 to 30,000 rpm. Terminator solutiontypically comprises 100 nL of a solution containing 5 picomoles of eachdeoxynucleotide, 0.5 picomoles of one dideoxynucleotide covalentlylinked to a fluorescent label, 90 mM Tris-HCl-(pH 7.5), 45 mM MgCl₂ and110 mM NaCl. The contents of the mixing chamber are transferred into areaction chamber containing 0.1 units of T7 DNA polymerase (or,alternatively, 0.1 Units of Taq polymerase) and 20 nL 0.1Mdithiothreitol (DTT) by spinning the disk at a rotational speed of 1 to30,000 rpm, yielding a reaction mixture in the reaction chamber having afinal concentration of buffer components that is 26 mM Tris-HCl (pH7.5), 13 mM MgCl 2, 32 mM NaCl, and 6 mM DTT. The reaction chamber isheated to 37° C. (or, alternatively, to 65° C. for Taq polymerase) by aresistive heating element integral to the disk, or alternatively,positioned within the device to specifically heat the reaction chamber,and incubated for a defined length of time, typically 1 minute. Thereaction products are spun into an equal volume of 90% formamide/EDTA,heated to 90° C. for 1 minute and spun to a capillary electrophoresisunit on the disk. The set of dideoxynucleotide-terminated DNA fragmentscomprising the reaction mixture is then separated by capillaryelectrophoresis and the sequence of fragments determined bylaser-induced fluorescence detection as described above. Diskscomprising a multiplicity of these synthetic arrays, permittingsimultaneous synthesis of a plurality of dideoxynucleotide-terminatedoligonucleotides, are also provided. The deducted nucleotide sequence isdetermined from the pattern of fluorescence signals detected and thesequence is determined from the pattern of fluorescence signals detectedand the sequence derived by the device microprocessor from these data.

EXAMPLE8 20 Liquid Phase Synthesis and Analysis

A variety of colorimetric chemical analyses are performed using a diskas described in Example 1. For example, a disk is provided (see FIG. 25)for performing a solution assay to determine iron concentration in atest solution (such as an industrial effluent) using a standardcolorimetric test. The device is fabricated with reagent reservoircontaining 40 uL 12N HCl, 100 uL 10% hydroxylamine hydrochloride, 100 uL10% sodium citrate buffer (pH 4), and 50 uL 0.02%, 1,10-phenanthroline.The reagent reservoirs are arranged as shown in FIG. 25 so that thesereagents are added to a reaction chamber sequentially by opening valvescontrolling flow from each reagent reservoir. Reagent transfer to thereaction chamber is achieved by spinning the disk of Example 1 at arotational speed of 1 to 30,000 rpm, whereby the centripetal forcemotivates each reagent solution from its reservoir to the reactionchamber. As shown in FIG. 25, sample is introduced through the sampleport (A) and centripetally delivered to the reaction chamber. The valveto the reagent reservoir containing HCl (B) is opened and acid is addedto the sample. The sample is incubated 10 minutes to dissolve all ironoxide present. Hydroxylamine hydrochloride (reservoir D) and citrate(reservoir E) are next added to the reaction mixture. The reactionmixture is incubated 20 minutes to ensure complete reduction of iron IIIto iron II. Next, 1,10-phenanthroline is transferred from reservoir F tocomplex the iron II and form a colored product. The solution isincubated 30 minutes at 30° C. to complete color development.Photometric measurement at 520 nm is done after the incubation processin a “read” cell (G) connected to the reaction chamber through valve G.

EXAMPLE 9 Solid Phase (Surface/Colloid) Synthesis/Analysis

Oligonucleotides, single-stranded DNA or duplex DNA is covalently linkedto a reactive particle (such as a bead or magnetic particle or achromatographic substrate) using a disk prepared as described in Example1 and shown in FIG. 26. In the illustrate embodiment, a 25 uL aliquot ofcarboxy-activated magnetic particles (BioMag 4125, PerSeptiveDiagnostics, Framingham, Mass.) is added to the disk through a sampleintroduction port. The particles are exchanged from the initial solutioninto 50 uL 0.1 M imidazole (pH 6) by decanting the original solutionthrough a valve to an effluent or waste reservoir, whereby the valve isconfigured to prevent loss of the magnetic particles from the reactionchamber. The imidazole solution is then added to the particle reactionchamber from an imidazole reservoir on the disk, transfer of imidazolebeing controlled by a valve. The motive force for both decanting theoriginal magnetic particle solution and transferring imidazole from theimidazole reservoir to the particle reaction chamber is provided byspinning the disk at a rotational speed of 1 to 30,000 rpm. Specificallywith reference to FIG. 26, as the disk spins the dense magneticparticles are pelleted in a funnel at the end of the reaction chamberand deposited to waste. A valve controlling an imidazole reagent chambercontaining 50 uL of 0.1M imidazole is then opened above the particlesbut below the decanting level and used to transfer the particles througha valve in the reaction chamber and into the next decanting reservoir.This decanting process can be repeated many times to affect a change inthe liquid phase to the desired composition. Typically, three exchangesare sufficient. Alternatively, appropriate configuration of the reagentand reaction chambers allows the magnetic particles to be exchangedwithin a single reaction chamber by controlled addition and removal ofimidazole from clusters of reagent reservoirs, or alternatively, asingle reagent reservoir large enough to contain sufficient imidazolefor the entire cycle of exchange.

After the exchange cycle is complete, the magnetic particles aretransferred to a next reaction chamber containing 250 ug dry1-ethyl-3(3-dimenthylaminopropyl) carbodiimide (EDAC). A reagentreservoir containing 170 OD (170 ng) 5′-aminated DNA oligonucleotide in50 uL of 0.1 M imidazole solution chamber prior to addition of theparticles in order to dissolve the EDAC. The particles are then addedthrough a valve in about 100 uL 0.1 M imidazole. Upon addition of themagnetic particles to the reaction chamber, the device is stopped andincubated 6 hours at 40° C. Heating can be effected by a heat source(such as Peltier heating device) embedded in the disk itself, orpositioned in the instrument in a configuration permitting specificheating of the reaction chamber. In the latter alternative, the disk maybe stopped at a predetermined position relative to the device to ensurespecificity of heating of the reaction chamber.

After incubation, the particles are washed and exchanged into 100 uLportions of water by decanting as described above as the disk is spun.Three exchanges are typically performed to purify the particles. Productis advantageously collected in the extremity of the disk where it caneasily be accessed for subsequent use. Disks comprising a multiplicityof these synthetic arrays, permitting simultaneous synthesis of aplurality of particle-linked oligonucleotides, are also provided.

EXAMPLE 10 Micro-Extraction System

A disk as described in Example 1 (see FIG. 27) for performingmicro-extraction of a solute from a solution or of a component of amixture as an alternative to HPLC or other conventional biochemicalseparation methodology. Specifically, a channel on the disk is coatedwith a compound (such as octanol) by standard procedures to provide asurface having an affinity for a component of a mixture, typically acomplex chemical or biochemical mixture. With a silicon disk, forexample, the surface of the channel is activated by filling the chamberwith aqueous epoxysilane at 95° C. for 1 hour. The disk is washed aboutfive times with distilled water to remove unreacted silane, andaminooctane is added in a solvent and incubated at 95° C. for 1 hourfollowed by solvent rinse to remove unreacted octane.

Sample mixture containing the component to be eluted is added to aninjection port and moved through the coated separation channel byrotating the disk at 1 to 30,000 rpm. Reagent reservoirs are opened atthe entrance of the channel and used to elute the sample retained on thecoated channel to a collection reservoir. The isolated sample componentis then collected at an outlet port.

EXAMPLE 11 Free Zone Capillary Electrophoresis

Free zone capillary electrophoresis is performed on a disk fabricated asdescribed in Example 1 above, and schematically represented in FIG. 28.Specifically, a 5 μm×75 μm×25 mm capilliary (it will be recognized thatall dimensions are approximate within limits of precision in fabricatingcomponents such as capillaries in the disk), is lithographically etchedonto a glass disk. Electrical connections are made using standardmethods by plating platinum onto the non-etched surface of the glassbefore sealing the top to the device. The separation channel isintersected by a 15 mm sample introduction channel, positioned 3 mm awayfrom a buffer reservoir. The interesting channel has a sample inlet portat one end and electrical connections at either end to control sampleapplication to the capillary.

In the practice of capillary electrophoresis on the disk, the separationchannel is filled from the buffer reservoir by rotation of the disk at aspeed of 1 to 30,000 rpm. Once the channel is filled, rotation isstopped until pressure needs to be applied to the channel again. Sampleis introduced by applying a voltage between the intersecting analyteinlet and analyte outlet channels on the chip (see FIG. 28) A 50 Vpotential drop is applied between the sample inlet and outlet portswhile the separation channel ports float. The sample, comprising asolution of 5 mM EDTA, 1 mM Tris-HC 1 (pH 8) with 1 mM Mg⁺² and 1 mMCa²⁺(typically prepared from the chloride salt). The running bufferconsists of 10 mM Tris-HCl (pH 8), 5 mM EDTA. Separation toward thecathode is then performed by floating the electric potential at thesample reservoir and applying 250 V along the separation channel.Separation is monitored at a position 2 cm from the inlet port bymonitoring, e.g. UV absorbance at 254 nm using a UV light source(mercury lamp) and a photodiode detector, positioned on the device tointerest the capillary channel.

EXAMPLE 12 DNA Electrophoresis

Gel electrophoresis is performed on a disk prepared as described inExample 1 above. For this application, a gel media is prepared in theseparation channel; however, such gel media must be protected from sheerforces that develop with rotation of the disk during transfer of sampleor buffer to the electrophoresis channel. Thus, the gel-filled capillaryis advantageously arrayed concentrically on the disk, as shownschematically in FIG. 29. As a result, the gel will only experienceshear forces from centripetal-induced pressure during rotation if afluid reservoir is in contact with the capillary during rotation of thedisk. At rest, the planar geometry of the disk prevents hydrodynamicpressure on the capillary. This is an advantage over standard capillaryelectrophoresis systems, where hydrodynamic pressure is not so easilycontrolled because the buffer volumes and reservoir heights need to becarefully adjusted before each run to avoid hydrodynamic flow. This isalso an advantage of capillary electrophoresis performed on the disks ofthe invention over electrophoresis performed on microchips, where bufferreservoirs are positioned above the plane of the separation channel andare thereby susceptible to hydrodynamic pressure-driven fluid flow. Gelelectrophoresis is performed on the disks of the invention to separateDNA fragments, including duplex PCR fragments, oligonucleotides andsingle-stranded, dideoxynucleotide-terminated enzymatic DNA sequencingcomponents, the system is configured as shown in FIG. 29. The disk isprepared comprising a polyacrylamide gel concentrically arrayed in amicroetched separation channel in the disk. The polyacrylamide gel isprepared from an unpolymerized solution of 7M urea, 45 mM Tris-boratebuffer (pH 8.3), 1 mM EDTA, 9% acrylamide, 0.1% TEMED and 10% ammoniumpersulfate. The disk can be prepared in the separation channel by mixingthe components (wherein it will be recognized that unpolymerizedpolymerized polyacrylamide is susceptible to light-catalyzedpolymerization upon storage) particularly by introducing TEMED andammonium persulfate to the mixture. Sufficient gel mixture is added tothe separation channel by opening a valve from a mixing chamber to theseparation channel and rotating the disk at 1 to 30,000 rpm. The disk isstopped upon filing of the separation channel to permit gelpolymerization. Shortly before polymerization is complete, the exitchannel is flushed to eliminate bubbles and unpolymerized monomer byflushing the channel with buffer from a large buffer reservoir at theoutlet side of the channel, controlled by a valve. A similar process isconducted on the inlet side of the gel.

To introduce a DNA sample, a valve is opened from an inlet port holdinga solution of DNA fragments, or alternatively, the sample is pipetteddirectly onto the disk. The sample is applied to the separation channelby spinning the disk at 1 to 30,000 rpm, forcing sample and buffer intothe buffer filled channel above the gel. Upon introduction of the sampleto the separation channel and the sample inlet channel. Sampleconcentrates at the gel/buffer interface before entering the separationmatrix, analogous to sample concentration during conventional slab gelelectrophoresis. Electrophoresis is performed at 250 V/cm to effect aseparation of DNA fragments, the cathode (positive electrode) beingpositioned at the outlet end of the channel distal to the sample inletchannel. A laser induced fluorescence detector is positioned at theoutlet of the gel filled capillary chamber to detect the labeled DNAfragments, as described above in Example 2.

EXAMPLE 13 Spectrophotometer Pathlength Extension

Spectrophotometric measurements in a rotating structure of the inventioncan be limited by the relatively small pathlengths provided byspectrophotometric illumination across the transverse dimension of thedisk. The intensity of absorbance of a solution is dependent on thedepth of the absorbing layer, as well as the concentration of theabsorbing molecules (as described in the Lambert-Beer law).

Although a measurement cell in a rotating microsystem platform of theinvention presents a short transverse pathlength, the lateral pathlengththrough the disk can be extensive (i.e., centimeters versusmillimeters). Spectral measurements can be enhanced by introducing lightthrough the detection chamber in the lateral dimension.

One arrangement providing transverse illumination in the lateraldimension is shown in FIG. 16. Light is beamed in a perpendiculardirection towards the disk. A mirror is positioned at a 45° angle to thedirection of the illuminating beam, whereby the light is directedlaterally through the detection chamber. Light passes through thedetection cell and is redirected by another 45° mirror onto aphotosensitive detector, such as a photodiode or photomultiplier tube.These mirrors can be inserted onto the disk, integrally molded into thedisk or metallicized in the plastic or other substrate comprising thedisk.

EXAMPLE 14 Cell Counting, Identification and Monitoring

Methods for identifying particular cells or cell types in a biologicalsample are provided. For example, a microplatform of the invention isprepared by having a surface adsorbly coated with monoclonal antibodyspecific to E. coli., the remaining sites being blocked with BSA. A milksample is introduced onto the disk and placed into contact with areaction chamber comprising the surface coated with the antibody. Themilk is incubated in this chamber for 30 min. The microsystem platformis then rotated to remove unwanted materials. An amount of a bufferappropriate for washing the microsystem chamber is then added to thesurface or chamber through a microchannel from a reservoir containingwashing buffer, said buffer being released by centrifugal force andopening of a microvalve. In a useful embodiment, the washing buffercomprises an E. coli-specific monoclonal antibody crosslinked to anenzyme (such peroxidase). Thus incubation is allowed to proceed for 5min. The disk is again spun with the opening of the appropriatemicrovalves to remove the washing solution from the chamber and to add asolution containing an enzymatic substrate (tetramethylbenzidine andhydrogen peroxide, maintained heretofore in a reagent reservoirconnected to the reaction chamber by a microvalve-controlledmicrochannel. The amount of E. coli bound in the reaction chamber isquantititated with regard to the amount of detected enzymatic activity,which is determined spectrophotometrically by the appearance of alight-absorbing product or the disappearance of a light-absorbingsubstrate.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention.

1. A method for performing assays using a microsystem platform, whereinthe microsystem platform comprises: a multiplicity of sample inlet portsarranged around the center of the microsystem platform for receiving abiological sample, wherein each of the sample inlet ports is operativelylinked to; a multiplicity of microchannels arrayed radially away fromthe center of the microsystem platform, said microchannels beingoperatively linked to; a multiplicity of reagent reservoirs containing areagent specific for an analyte to be measured, wherein release of thereagent from each of the reservoirs is controlled by a microvalve, andwherein the multiplicity of microchannels is also operatively linked to;a multiplicity of analyte detection chambers arranged peripherallyaround the outer edge of the microsystem platform, wherein movement ofthe biological sample from the inlet port and through the microchanneland movement of the reagent from the reagent reservoir and through themicrochannel is motivated by centripetal force generated by rotationalmotion of the microsystem platform, the method comprising: operatingeach of the microvalves to control release of the reagent from thereagent reservoirs by generating a signal, at a time and for a durationwhereby the reagent moves into the microchannel and is mixed with thebiological sample; detecting an amount of analyte present in thebiological sample; and storing data representing the amount of analytepresent in the biological sample upon the microsystem platform.
 2. Themethod of claim 1, wherein operating each of the microvalves to controlrelease of the reagent from the reagent reservoirs by generating asignal comprises: setting an RPM for a first valve actuation; and uponreaching the RPM, spinning the microsystem platform at the RPM.
 3. Themethod of claim 2, further comprising setting an RPM for a second valveactuation.
 4. The method of claim 1, further comprising outputting arepresentation of the amount of analyte present in the biologicalsample.
 5. The method of claim 4, wherein outputting the representationcomprises sending the representation to a display.
 6. The method ofclaim 1, further comprising identifying a status of the microvalveoperation.
 7. The method of claim 6, further comprising if the status isunacceptable, terminating the method.
 8. The method of claim 1, furthercomprising sending the data representing the amount of analyte presentin the biological sample to a storage device via communication selectedfrom the group consisting of telephone, facsimile transmission, andwireless communication.
 9. The method of claim 1, wherein operating eachof the microvalves to control release of the reagent from the reagentreservoirs by generating a signal comprises calculating the timerequired to transfer the reagent through the microchannel to mix withthe biological sample.
 10. The method of claim 9, wherein calculatingthe time required to transfer the reagent through the microchannel tomix with the biological sample comprises calculating D_(t) by:D _(t) =V/Q, if L≦(4V/πD ²), andD _(t)=(V/Q)*(4πD ² L/4V), if L>(4V/πD ²) wherein D_(t) is the timerequired to transfer a volume V from a reservoir through a microchannelof length L, Q is a rate of flow, and D is a diameter of themicrochannel.
 11. A computer readable medium having stored thereininstructions for causing a processing unit to execute the method ofclaim
 1. 12. A system comprising: a microsystem platform including: amultiplicity of sample inlet ports arranged around the center of themicrosystem platform for receiving a biological sample, wherein each ofthe sample inlet ports is operatively linked to; a multiplicity ofmicrochannels arrayed radially away from the center of the microsystemplatform, said microchannels being operatively linked to; a multiplicityof reagent reservoirs containing a reagent specific for an analyte to bemeasured, wherein release of the reagent from each of the reservoirs iscontrolled by a microvalve, wherein movement of the biological samplefrom the inlet port and through the microchannel and movement of thereagent from the reagent reservoir and through the microchannel ismotivated by centripetal force generated by rotational motion of themicrosystem platform, a processing unit; and machine languageinstructions stored in data storage executable by the processing unit toperform functions including: operating each of the microvalves tocontrol release of the reagent from the reagent reservoirs by generatinga signal, at a time and for a duration whereby the reagent moves intothe microchannel and is mixed with the biological sample; detecting anamount of analyte present in the biological sample; and storing datarepresenting the amount of analyte present in the biological sample uponthe microsystem platform.
 13. The system of claim 12, wherein themachine language instructions are executable to further performfunctions including: generating the time by calculatingD _(t) =V/Q, if L≦(4V/πD ²), andDt=(V/Q)*(4πD ² L/4V), if L>(4V/TD ²) wherein D_(t) is the time requiredto transfer a volume V from a reservoir through a microchannel of lengthL, Q is a rate of flow, and D is a diameter of the microchannel.